PUFA Polyketide Synthase Systems and Uses Thereof

ABSTRACT

Disclosed are the complete polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from  Schizochytrium , and biologically active fragments and homologues thereof. More particularly, this invention relates to nucleic acids encoding such PUFA PKS systems, to proteins and domains thereof that comprise such PUFA PKS systems, to genetically modified organisms (plants and microorganisms) comprising such PUFA PKS systems, and to methods of making and using the PUFA PKS systems disclosed herein. This invention also relates to genetically modified plants and microorganisms and methods to efficiently produce lipids enriched in various polyunsaturated fatty acids (PUFAs) as well as other bioactive molecules by manipulation of a PUFA polyketide synthase (PKS) system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.11/452,138, filed Jun. 12, 2006, which claims the benefit of priorityunder 35 U.S.C. §119(e) from U.S. Provisional Application No.60/784,616, filed Mar. 21, 2006, and from U.S. Provisional ApplicationNo. 60/689,167, filed Jun. 10, 2005. U.S. application Ser. No.11/452,138 is also a continuation-in-part of U.S. patent applicationSer. No. 10/124,800, filed Apr. 16, 2002, which claims priority under 35U.S.C. §119(e) from: U.S. Provisional Application Ser. No. 60/284,066,filed Apr. 16, 2001; U.S. Provisional Application Ser. No. 60/298,796,filed Jun. 15, 2001; and U.S. Provisional Application Ser. No.60/323,269, filed Sep. 18, 2001. U.S. patent application Ser. No.10/124,800 is also a continuation-in-part of U.S. application Ser. No.09/231,899, filed Jan. 14, 1999, now U.S. Pat. No. 6,566,583. Each ofthe above-identified patent applications is incorporated herein byreference in its entirety for all purposes.

U.S. application Ser. No. 11/452,138 does not claim the benefit ofpriority from U.S. application Ser. No. 09/090,793, filed Jun. 4, 1998,now U.S. Pat. No. 6,140,486, although U.S. application Ser. No.09/090,793 is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted on a compactdisc, in duplicate. Each of the two compact discs, which are identicalto each other pursuant to 37 CFR §1.52(e)(4), contains the followingfile: “Sequence Listing”, having a size in bytes of 301 KB, recorded on12 Jun. 2006. The information contained on the compact disc is herebyincorporated by reference in its entirety pursuant to 37 CFR§1.77(b)(4).

FIELD OF THE INVENTION

This invention relates to polyunsaturated fatty acid (PUFA) polyketidesynthase (PKS) systems from Schizochytrium. More particularly, thisinvention relates to nucleic acids encoding such PUFA PKS systems, tosuch PUFA PKS systems, to genetically modified organisms comprising suchPUFA PKS systems, and to methods of making and using such PUFA PKSsystems disclosed herein. This invention also relates to PUFA PKSsystems from non-bacterial and bacterial organisms identified using theSchizochytrium PUFA PKS systems described herein.

BACKGROUND OF THE INVENTION

Polyketide synthase (PKS) systems are generally known in the art asenzyme complexes related to fatty acid synthase (FAS) systems, but whichare often highly modified to produce specialized products that typicallyshow little resemblance to fatty acids. It has now been shown, however,that polyketide synthase systems exist in marine bacteria and certaineukaryotic organisms that are capable of synthesizing polyunsaturatedfatty acids (PUFAs) from acetyl-CoA and malonyl-CoA. The PKS pathwaysfor PUFA synthesis in Shewanella and another marine bacteria, Vibriomarinus, are described in detail in U.S. Pat. No. 6,140,486. The PKSpathways for PUFA synthesis in the eukaryotic Thraustochytrid,Schizochytrium is described in detail in U.S. Pat. No. 6,566,583. ThePKS pathways for PUFA synthesis in eukaryotes such as members ofThraustochytriales, including the structural description of a PUFA PKSsystem in Schizochytrium and the identification of a PUFA PKS system inThraustochytrium, including details regarding uses of these systems, aredescribed in detail in U.S. Patent Application Publication No.20020194641, published Dec. 19, 2002 (corresponding to U.S. patentapplication Ser. No. 10/124,800, filed Apr. 16, 2002). U.S. PatentApplication Publication No. 20040235127, published Nov. 25, 2004(corresponding to U.S. patent application Ser. No. 10/810,352, filedMar. 24, 2004), discloses the structural description of a PUFA PKSsystem in Thraustochytrium, and further detail regarding the productionof eicosapentaenoic acid (C20:5, ω-3) (EPA) and other PUFAs using suchsystems. U.S. Patent Application Publication No. 20050100995, publishedMay 12, 2005 (corresponding to U.S. patent application Ser. No.10/965,017, filed Oct. 13, 2004), discloses the structural andfunctional description of PUFA PKS systems in Shewanella olleyana andShewanella japonica, and uses of such systems. These applications alsodisclose the genetic modification of organisms, including microorganismsand plants, with the genes comprising the PUFA PKS pathway and theproduction of PUFAs by such organisms. Furthermore, PCT PatentPublication No. WO 05/097982 describes a PUFA PKS system in Ulkenia,U.S. Patent Application Publication No. 20050014231 describes PUFA PKSgenes and proteins from Thraustochytrium aureum.

Researchers have attempted to exploit polyketide synthase (PKS) systemsthat have been traditionally described in the literature as falling intoone of three basic types, typically referred to as: Type I (modular oriterative), Type II, and Type III. For purposes of clarity, it is notedthat the Type I modular PKS system has previously also been referred toas simply a “modular” PKS system, and the Type I iterative PKS systemhas previously also been referred to simply as a “Type I” PKS system.The Type II system is characterized by separable proteins, each of whichcarries out a distinct enzymatic reaction. The enzymes work in concertto produce the end product and each individual enzyme of the systemtypically participates several times in the production of the endproduct. This type of system operates in a manner analogous to the fattyacid synthase (FAS) systems found in plants and bacteria. Type Iiterative PKS systems are similar to the Type II system in that theenzymes are used in an iterative fashion to produce the end product. TheType I iterative differs from Type II in that enzymatic activities,instead of being associated with separable proteins, occur as domains oflarger proteins. This system is analogous to the Type I FAS systemsfound in animals and fungi.

In contrast to the Type II systems, in Type I modular PKS systems, eachenzyme domain is used only once in the production of the end product.The domains are found in very large proteins and the product of eachreaction is passed on to another domain in the PKS protein.Additionally, in the PKS systems described above, if a carbon-carbondouble bond is incorporated into the end product, it is usually in thetrans configuration.

Type III systems have been more recently discovered and belong to theplant chalcone synthase family of condensing enzymes. Type III PKSs aredistinct from type I and type II PKS systems and utilize free CoAsubstrates in iterative condensation reactions to usually produce aheterocyclic end product.

Polyunsaturated fatty acids (PUFAs) are considered to be useful fornutritional, pharmaceutical, industrial, and other purposes. The currentsupply of PUFAs from natural sources and from chemical synthesis is notsufficient for commercial needs. A major current source for PUFAs isfrom marine fish; however, fish stocks are declining, and this may notbe a sustainable resource. Additionally, contamination, from both heavymetals and toxic organic molecules, is a serious issue with oil derivedfrom marine fish. Vegetable oils derived from oil seed crops arerelatively inexpensive and do not have the contamination issuesassociated with fish oils. However, the PUFAs found in commerciallydeveloped plant oils are typically limited to linoleic acid (eighteencarbons with 2 double bonds, in the delta 9 and 12 positions—18:2 delta9,12) and linolenic acid (18:3 delta 9,12,15). In the conventionalpathway (i.e., the “standard” pathway or “classical” pathway) for PUFAsynthesis, medium chain-length saturated fatty acids (products of afatty acid synthase (FAS) system) are modified by a series of elongationand desaturation reactions. The substrates for the elongation reactionare fatty acyl-CoA (the fatty acid chain to be elongated) andmalonyl-CoA (the source of the 2 carbons added during each elongationreaction). The product of the elongase reaction is a fatty acyl-CoA thathas two additional carbons in the linear chain. The desaturases createcis double bonds in the preexisting fatty acid chain by extraction of 2hydrogens in an oxygen-dependant reaction. The substrates for thedesaturases are either acyl-CoA (in some animals) or the fatty acid thatis esterified to the glycerol backbone of a phospholipid (e.g.phosphatidylcholine).

Therefore, because a number of separate desaturase and elongase enzymesare required for fatty acid synthesis from linoleic and linolenic acidsto produce the more unsaturated and longer chain PUFAs, engineeringplant host cells for the expression of PUFAs such as EPA anddocosahexaenoic acid (DHA) may require expression of several separateenzymes to achieve synthesis. Additionally, for production of useablequantities of such PUFAs, additional engineering efforts may berequired. Therefore, it is of interest to obtain genetic materialinvolved in PUPA biosynthesis from species that naturally produce thesefatty acids (e.g., from a PUFA PKS system) and to express the isolatedmaterial alone or in combination in a heterologous system which can bemanipulated to allow production of commercial quantities of PUFAs.

There have been many efforts to produce PUFAs in oil-seed crop plants bymodification of the endogenously-produced fatty acids. Geneticmodification of these plants with various individual genes for fattyacid elongases and desaturases has produced leaves or seeds containingmeasurable levels of PUFAs such as EPA, but also containing significantlevels of mixed shorter-chain and less unsaturated PUFAs (Qi et al.,Nature Biotech. 22:739 (2004); PCT Publication No. WO 04/071467; Abbadiet al., Plant Cell 16:1 (2004)); Napier and Sayanova, Proceedings of theNutrition Society (2005), 64:387-393; Robert et al., Functional PlantBiology (2005) 32:473-479; or U.S. Patent Application Publication2004/0172682.

Improvement in both microbial and plant production of PUFAs is a highlydesirable commercial goal. Therefore, there remains a need in the artfor a method to efficiently and effectively produce quantities of lipids(e.g., triacylglycerol (TAG) and phospholipid (PL)) enriched in desiredPUFAs, particularly in commercially useful organisms such asmicroorganisms and oil-seed plants.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an isolated nucleicacid molecule comprising a nucleic acid sequence selected from: (a) anucleic acid sequence selected from: SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5; (b) a nucleic acid sequence encoding an amino acid sequenceselected from: SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; (c) a nucleicacid sequence encoding an amino acid sequence that is at least 90%identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, whereinsaid amino acid sequence has β-keto acyl-ACP synthase (KS) activity,malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein(ACP) activity and ketoreductase (KR) activity, and wherein said aminoacid sequence comprises an aspartate at a position corresponding toamino acid 667 of SEQ ID NO:2 and a histidine at a positioncorresponding to amino acid 668 of SEQ ID NO:2; (d) a nucleic acidsequence encoding an amino acid sequence that is at least 90% identicalto SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said aminoacid sequence has KS activity, chain length factor (CLF) activity, acyltransferase (AT) activity, and enoyl ACP-reductase (ER) activity, andwherein said amino acid sequence comprises a valine at a positioncorresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at aposition corresponding to amino acid 1415 of SEQ ID NO:4; and (e) anucleic acid sequence encoding an amino acid sequence that is at least90% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6,wherein said amino acid sequence has FabA-like β-hydroxy acyl-ACPdehydrase (DH) activity and ER activity, and wherein said amino acidsequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I(positions 876-890 of SEQ ID NO:6) at positions corresponding to aminoacids 876-890 of SEQ ID NO:6.

In one aspect, the nucleic acid molecule comprising a nucleic acidsequence selected from: (a) a nucleic acid sequence encoding an aminoacid sequence that is at least 95% identical to SEQ ID NO:2 or that is afragment of SEQ ID NO:2, wherein said amino acid sequence has β-ketoacyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT)activity, acyl carrier protein (ACP) activity and ketoreductase (KR)activity, and wherein said amino acid sequence comprises an aspartate ata position corresponding to amino acid 667 of SEQ ID NO:2 and ahistidine at a position corresponding to amino acid 668 of SEQ ID NO:2;(b) a nucleic acid sequence encoding an amino acid sequence that is atleast 95% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4,wherein said amino acid sequence has KS activity, chain length factor(CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase(ER) activity, and wherein said amino acid sequence comprises a valineat a position corresponding to amino acid 371 of SEQ ID NO:4 and aglutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4;and (c) a nucleic acid sequence encoding an amino acid sequence that isat least 95% identical to SEQ ID NO:6 or that is a fragment of SEQ IDNO:6, wherein said amino acid sequence has FabA-like β-hydroxy acyl-ACPdehydrase (DH) activity and ER activity, and wherein said amino acidsequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I(positions 876-890 of SEQ ID NO:6) at positions corresponding to aminoacids 876-890 of SEQ ID NO:6.

In one aspect, the nucleic acid molecule comprises a nucleic acidsequence encoding an amino acid sequence selected from SEQ ID NO:2, SEQID NO:4 and SEQ ID NO:6. In another aspect, the nucleic acid moleculecomprises a nucleic acid sequence selected from: SEQ ID NO:1, SEQ IDNO:3, and SEQ ID NO:5.

In one aspect of this embodiment, the nucleic acid molecule of (a)comprises a nucleic acid sequence encoding the amino acid sequenceencoded by a plasmid selected from: pKJ1126 (ATCC Accession No. ______),pJK306 (ATCC Accession No. ______), and pJK320 (ATCC Accession No.______). In one aspect of this embodiment, the nucleic acid molecule of(b) comprises a nucleic acid sequence encoding the amino acid sequenceencoded by a plasmid selected from: pJK1129 (ATCC Accession No. ______),and pJK324 (ATCC Accession No. ______). In another aspect of thisembodiment, the nucleic acid molecule of (c) comprises a nucleic acidsequence encoding the amino acid sequence encoded by a plasmid selectedfrom: pJK1131 (ATCC Accession No. ______) and pBR002 (ATCC Accession No.______).

Another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected from:(a) a first nucleic acid sequence encoding a first amino acid sequencethat has β-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACPacyltransferase (MAT) activity, acyl carrier protein (ACP) activity andketoreductase (KR) activity, wherein the first nucleic acid sequencehybridizes under very high stringency conditions to the complement of asecond nucleic acid sequence encoding a second amino acid sequence ofSEQ ID NO:2, and wherein said first amino acid sequence comprises anaspartate at a position corresponding to amino acid 667 of SEQ ID NO:2and a histidine at a position corresponding to amino acid 668 of SEQ IDNO:2; (b) a first nucleic acid sequence encoding a first amino acidsequence that has KS activity, chain length factor (CLF) activity, acyltransferase (AT) activity, and enoyl ACP-reductase (ER) activity,wherein the first nucleic acid sequence hybridizes under very highstringency conditions to the complement of a second nucleic acidsequence encoding a second amino acid sequence of SEQ ID NO:4, andwherein said first amino acid sequence comprises a valine at a positioncorresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at aposition corresponding to amino acid 1415 of SEQ ID NO:4; and (c) afirst nucleic acid sequence encoding a first amino acid sequence thathas FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity and ERactivity, wherein the first nucleic acid sequence hybridizes under veryhigh stringency conditions to the complement of a second nucleic acidsequence encoding a second amino acid sequence of SEQ ID NO:6, andwherein said first amino acid sequence comprises the sequence ofH-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) atpositions corresponding to amino acids 876-890 of SEQ ID NO:6. In oneaspect of this embodiment, the first nucleic acid sequence is isolatedfrom a Schizochytrium, such as, but not limited to, Schizochytrium ATCC20888.

Yet another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected from:(a) a nucleic acid sequence of SEQ ID NO:9; (b) a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:10; and (c) a nucleic acidsequence encoding an amino acid sequence that is at least 90% identicalto SEQ ID NO:10 or that is a fragment of SEQ ID NO:10, wherein the aminoacid sequence has malonyl-CoA:ACP acyltransferase (MAT) activity, andwherein said amino acid sequence comprises an aspartate at a positioncorresponding to amino acid 93 of SEQ ID NO:10 and a histidine at aposition corresponding to amino acid 94 of SEQ ID NO:10. In one aspectof this embodiment, the nucleic acid molecule comprises a nucleic acidsequence encoding an amino acid sequence that is at least 95% identicalto SEQ ID NO:10 or that is a fragment of SEQ ID NO:10, wherein the aminoacid sequence has malonyl-CoA:ACP acyltransferase (MAT) activity, andwherein said amino acid sequence comprises an aspartate at a positioncorresponding to amino acid 93 of SEQ ID NO:10 and a histidine at aposition corresponding to amino acid 94 of SEQ ID NO:10. In one aspect,the nucleic acid molecule comprises a nucleic acid sequence encoding anamino acid sequence of SEQ ID NO:10.

Another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected from:(a) a nucleic acid sequence of SEQ ID NO:19; (b) a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:20; and (c) a nucleic acidsequence encoding an amino acid sequence that is at least 90% identicalto SEQ ID NO:20 or that is a fragment of SEQ ID NO:20, wherein the aminoacid sequence has β-keto acyl-ACP synthase (KS) activity, and whereinsaid amino acid sequence comprises a valine at a position correspondingto amino acid 371 of SEQ ID NO:20. In one aspect of this embodiment, thenucleic acid molecule comprises a nucleic acid sequence encoding anamino acid sequence that is at least 95% identical to SEQ ID NO:20 orthat is a fragment of SEQ ID NO:20, wherein the amino acid sequence hasβ-keto acyl-ACP synthase (KS) activity, and wherein said amino acidsequence comprises a valine at a position corresponding to amino acid371 of SEQ ID NO:20. In one aspect, the nucleic acid molecule comprisesa nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:20.

Yet another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected from:(a) a nucleic acid sequence of SEQ ID NO:29; (b) a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:30; and (c) a nucleic acidsequence encoding an amino acid sequence that is at least 90% identicalto SEQ ID NO:30 or that is a fragment of SEQ ID NO:30, wherein the aminoacid sequence has FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity,and wherein said amino acid sequence comprises the sequence ofH-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) atpositions corresponding to amino acids 426-440 of SEQ ID NO:30. In oneaspect, the nucleic acid molecule comprises a nucleic acid sequenceencoding an amino acid sequence that is at least 95% identical to SEQ IDNO:30 or that is a fragment of SEQ ID NO:30, wherein the amino acidsequence has FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity, andwherein said amino acid sequence comprises the sequence ofH-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) atpositions corresponding to amino acids 426-440 of SEQ ID NO:30. In oneaspect, the nucleic acid molecule comprises a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:30.

Another embodiment of the invention relates to a recombinant nucleicacid molecule comprising any of the nucleic acid molecules describedabove, operatively linked to at least one transcription controlsequence.

Yet another embodiment of the invention relates to a recombinant celltransfected with any of the nucleic acid molecules described above. Inone aspect, the recombinant cell is a microorganism. In another aspect,the recombinant cell is a plant cell.

Another embodiment of the present invention relates to an isolatednucleic acid molecule consisting essentially of a nucleic acid sequencethat is fully complementary to any of the nucleic acid moleculesdescribed above.

Another embodiment of the present invention relates to a geneticallymodified microorganism that has been transformed with any of the nucleicacid molecules described above.

Yet another embodiment of the present invention relates to a geneticallymodified plant that has been transformed with any of the nucleic acidmolecules described above.

Another embodiment of the present invention relates to a geneticallymodified microorganism that has been transformed with: (a) a nucleicacid molecule comprising a nucleic acid sequence encoding an amino acidsequence that is at least 90% identical to SEQ ID NO:2 or that is afragment of SEQ ID NO:2, wherein said amino acid sequence has β-ketoacyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT)activity, acyl carrier protein (ACP) activity and ketoreductase (KR)activity, and wherein said amino acid sequence comprises an aspartate ata position corresponding to amino acid 667 of SEQ ID NO:2 and ahistidine at a position corresponding to amino acid 668 of SEQ ID NO:2;(b) a nucleic acid molecule comprising a nucleic acid sequence encodingan amino acid sequence that is at least 90% identical to SEQ ID NO:4 orthat is a fragment of SEQ ID NO:4, wherein said amino acid sequence hasKS activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity, and wherein said aminoacid sequence comprises a valine at a position corresponding to aminoacid 371 of SEQ ID NO:4 and a glutamate at a position corresponding toamino acid 1415 of SEQ ID NO:4; and (c) a nucleic acid moleculecomprising a nucleic acid sequence encoding an amino acid sequence thatis at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ IDNO:6, wherein said amino acid sequence has FabA-like β-hydroxy acyl-ACPdehydrase (DH) activity and ER activity, and wherein said amino acidsequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I(positions 876-890 of SEQ ID NO:6) at positions corresponding to aminoacids 876-890 of SEQ ID NO:6. In one aspect, the microorganism has beentransformed with a nucleic acid molecule comprising a nucleic acidsequence encoding SEQ ID NO:2, a nucleic acid molecule comprising anucleic acid sequence encoding SEQ ID NO:4, and a nucleic acid moleculecomprising a nucleic acid sequence encoding SEQ ID NO:6. In one aspect,the microorganism endogenously expresses a PUFA PKS system. In anotheraspect, the microorganism has been further transformed with arecombinant nucleic acid molecule encoding a phosphopantetheinetransferase. The microorganism can include, but is not limited to, aThraustochytriales microorganism, a bacterium or a yeast.

Yet another embodiment of the present invention relates to a method toproduce a bioactive molecule, comprising culturing under conditionseffective to produce said bioactive molecule a genetically modifiedorganism described above. In one aspect, the bioactive molecule is apolyunsaturated fatty acid (PUFA).

Another embodiment of the present invention relates to a geneticallymodified plant or part of the plant, wherein said plant has beentransformed with: (a) a nucleic acid molecule comprising a nucleic acidsequence encoding an amino acid sequence that is at least 90% identicalto SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said aminoacid sequence has β-keto acyl-ACP synthase (KS) activity,malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein(ACP) activity and ketoreductase (KR) activity, and wherein said aminoacid sequence comprises an aspartate at a position corresponding toamino acid 667 of SEQ ID NO:2 and a histidine at a positioncorresponding to amino acid 668 of SEQ ID NO:2; (b) a nucleic acidmolecule comprising a nucleic acid sequence encoding an amino acidsequence that is at least 90% identical to SEQ ID NO:4 or that is afragment of SEQ ID NO:4, wherein said amino acid sequence has KSactivity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity, and wherein said aminoacid sequence comprises a valine at a position corresponding to aminoacid 371 of SEQ ID NO:4 and a glutamate at a position corresponding toamino acid 1415 of SEQ ID NO:4; and (c) a nucleic acid moleculecomprising a nucleic acid sequence encoding an amino acid sequence thatis at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ IDNO:6, wherein said amino acid sequence has FabA-like β-hydroxy acyl-ACPdehydrase (DH) activity and ER activity, and wherein said amino acidsequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I(positions 876-890 of SEQ ID NO:6) at positions corresponding to aminoacids 876-890 of SEQ ID NO:6. In one aspect, the plant has been furthergenetically modified to express a recombinant nucleic acid moleculeencoding a phosphopantetheine transferase. In one aspect, the plant is adicotyledonous plant, and in another aspect, the plant is amonocotyledonous plant. In another aspect, the plant is selected from:canola, soybean, rapeseed, linseed, corn, safflower, sunflower andtobacco.

In one aspect, the plant is an oilseed plant and the part of the plantis a mature oilseed. In one aspect, the total fatty acid profile in theplant or part of the plant comprises at least about 0.5% by weight of atleast one PUFA selected from DHA (docosahexaenoic acid (C22:6, n-3)) andDPA (docosapentaenoic acid (C22:5, n-6), and wherein the total fattyacids produced as a result of transformation with said nucleic acidmolecules, other than said at least one PUFA, comprise less than about10% of the total fatty acids produced by said plant. In one aspect, thetotal fatty acids produced as a result of transformation with saidnucleic acid molecules, other than said at least one PUFA, comprise lessthan 5% by weight of the total fatty acids produced by said plant. Inanother aspect, the fatty acids consisting of gamma-linolenic acid (GLA;18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds,PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAshaving 22 carbons and two or three carbon-carbon double bonds, compriseless than 5% by weight of the total fatty acids produced by said plant.In another aspect, gamma-linolenic acid (GLA; 18:3, n-6) comprises lessthan 1% by weight of the total fatty acids produced by said plant.

Yet another embodiment of the present invention relates to a plant or apart of the plant, wherein the total fatty acid profile in the plant orpart of the plant comprises detectable amounts of DHA (docosahexaenoicacid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), whereinthe ratio of DPAn-6 to DHA is 1:1 or greater than 1:1.

Another embodiment of the present invention relates to a plant or a partof the plant, wherein the total fatty acid profile in the plant or partof the plant comprises detectable amounts of DHA (docosahexaenoic acid(C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), wherein theratio of DPAn-6 to DHA is less than 1:1. In either of the twoembodiments above, in one aspect, the total fatty acid profile in theplant or part of the plant contains less than 5% by weight in total ofall of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAshaving 18 carbons and four carbon-carbon double bonds, PUFAs having 20carbons and three carbon-carbon double bonds, and PUFAs having 22carbons and two or three carbon-carbon double bonds.

Yet another embodiment of the present invention relates to plant or apart of the plant, wherein the total fatty acid profile in the plant orpart of the plant comprises at least about 0.5% by weight of at leastone polyunsaturated fatty acid (PUPA) selected from DHA (C22:6n-3) andDPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plantor part of the plant contains less than 5% in total of all of thefollowing PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18carbons and four carbon-carbon double bonds, PUFAs having 20 carbons andthree carbon-carbon double bonds, and PUFAs having 22 carbons and two orthree carbon-carbon double bonds.

Another embodiment of the present invention relates to a plant or a partof the plant, wherein the total fatty acid profile in the plant or partof the plant comprises at least about 0.5% by weight of at least onepolyunsaturated fatty acid (PUFA) selected from DHA (C22:6n-3) andDPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plantor part of the plant contains less than 1% of each of the followingPUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbonsand four carbon-carbon double bonds, PUFAs having 20 carbons and threecarbon-carbon double bonds, and PUFAs having 22 carbons and two or threecarbon-carbon double bonds.

Another embodiment of the present invention relates to a plant or a partof the plant, wherein the total fatty acid profile in the plant or partof the plant comprises at least about 0.5% by weight of at least onepolyunsaturated fatty acid (PUFA) selected from DHA (C22:6n-3) andDPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plantor part of the plant contains less than 2% of gamma-linolenic acid (GLA;18:3, n-6) and dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6).

Another embodiment of the present invention relates to seeds obtainedfrom any of the plants or part of plants described above, a food productcomprising such seeds, an oil obtained from such seeds, and a foodproduct comprising such oil. Also included in the invention is an oilblend comprising such oil and another oil, such as, but not limited to,a microbial oil, a fish oil, and a vegetable oil.

Yet another embodiment of the present invention relates to an oilcomprising the following fatty acids: DHA (C22:6n-3), DPAn-6 (C22:5n-6),oleic acid (C18:1), linolenic acid (C18:3), linoleic acid (C18:2),C16:0, C18.0, C20:0, C20:1n-9, C20:2n-6, C22:1n-9; wherein the oilcomprises less than 0.5% of any of the following fatty acids:gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and fourcarbon-carbon double bonds, PUFAs having 20 carbons and threecarbon-carbon double bonds, and PUFAs having 22 carbons and two or threecarbon-carbon double bonds.

Another embodiment of the present invention relates to an oilseed plantthat produces mature seeds in which the total seed fatty acid profilecomprises at least 1.0% by weight of at least one polyunsaturated fattyacid selected from DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein thetotal fatty acid profile in the plant or part of the plant contains lessthan 5% in total of all of the following PUFAs: gamma-linolenic acid(GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon doublebonds, PUFAs having 20 carbons and three carbon-carbon double bonds, andPUFAs having 22 carbons and two or three carbon-carbon double bonds.

Yet another embodiment of the present invention relates to an oilseedplant that produces mature seeds in which the total seed fatty acidprofile comprises at least 1.0% by weight of at least onepolyunsaturated fatty acid (PUFA) selected from DHA (C22:6n-3) andDPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plantor part of the plant contains less than 1% of gamma-linolenic acid (GLA;18:3, n-6).

Another embodiment of the present invention relates to a method toproduce a bioactive molecule, comprising growing under conditionseffective to produce said bioactive molecule a genetically modifiedplant as described above. In one aspect, the bioactive molecule is apolyunsaturated fatty acid (PUFA).

Yet another embodiment of the present invention relates to a method toproduce a plant that has a polyunsaturated fatty acid (PUFA) profilethat differs from the naturally occurring plant, comprising geneticallymodifying said plant to express a PUFA PKS system comprising at leastone of any of the nucleic acid molecules as described above.

Another embodiment of the present invention relates to a method toproduce a recombinant microbe, comprising genetically modifyingmicrobial cells to express at least one of any of the nucleic acidmolecules as described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the domain structure of theSchizochytrium PUFA PKS system.

FIG. 2 shows a comparison of PKS domains from Schizochytrium andShewanella.

FIG. 3 shows a GC FAME profile of control yeast and yeast expressingOrfs sA, sB, C and Het I.

FIG. 4 shows a GC FAME profile of the PUFA region from FIG. 3.

FIG. 5 shows GC FAME profiles of wild-type Arabidopsis and ArabidopsisLine 269 (plastid targeted).

FIG. 6 is a schematic diagram showing the construction of pSBS4107:Acyl-ACP transit peptide-HetI: Acyl-ACP transit peptide-ORFC.

FIG. 7 is a schematic diagram showing the construction of pSBS5720:Acyl-ACP transit peptide-ORFB.

FIG. 8 is a schematic diagram showing the construction of pSBS4757:Acyl-ACP transit peptide-ORFA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to polyunsaturated fatty acid(PUFA) polyketide synthase (PKS) systems from Schizochytrium, togenetically modified organisms comprising Schizochytrium PUFA PKSsystems, to methods of making and using such systems for the productionof products of interest, including bioactive molecules, and to PUFA PKSsystems identified using the structural information for theSchizochytrium PUFA PKS systems disclosed herein. In one preferredembodiment, the present invention relates to a method to produce PUFAsin an oil-seed plant that has been genetically modified to express aPUFA PKS system of the present invention. The oils produced by the plantcontain at least one PUFA produced by the PUFA PKS system and aresubstantially free of the mixed shorter-chain and less unsaturated PUFAsthat are fatty acid products produced by the modification of products ofthe FAS system.

As used herein, a PUFA PKS system (which may also be referred to as aPUFA synthase system or PUFA synthase) generally has the followingidentifying features: (1) it produces PUFAs, and particularly, longchain PUFAs, as a natural product of the system; and (2) it comprisesseveral multifunctional proteins assembled into a complex that conductsboth iterative processing of the fatty acid chain as well non-iterativeprocessing, including trans-cis isomerization and enoyl reductionreactions in selected cycles. In addition, the ACP domains present inthe PUFA synthase enzymes require activation by attachment of a cofactor(4-phosphopantetheine). Attachment of this cofactor is carried out byphosphopantetheinyl transferases (PPTase). If the endogenous PPTases ofthe host organism are incapable of activating the PUFA synthase ACPdomains, then it is necessary to provide a PPTase that is capable ofcarrying out that function. The inventors have identified the Het Ienzyme of Nostoc sp. as an exemplary and suitable PPTase for activatingPUFA synthase ACP domains. Reference to a PUFA PKS system or a PUFAsynthase refers collectively to all of the genes and their encodedproducts that work in a complex to produce PUFAs in an organism.Therefore, the PUFA PKS system refers specifically to a PKS system forwhich the natural products are PUFAs.

More specifically, first, a PUFA PKS system that forms the basis of thisinvention produces polyunsaturated fatty acids (PUFAs) and particularly,long chain PUFAs, as products (e.g., an organism that endogenously(naturally) contains such a PKS system makes PUFAs using this system).According to the present invention, PUFAs are fatty acids with a carbonchain length of at least 16 carbons, and more preferably at least 18carbons, and more preferably at least 20 carbons, and more preferably 22or more carbons, with at least 3 or more double bonds, and preferably 4or more, and more preferably 5 or more, and even more preferably 6 ormore double bonds, wherein all double bonds are in the cisconfiguration. Reference to long chain polyunsaturated fatty acids(LCPUFAs) herein more particularly refers to fatty acids of 18 and morecarbon chain length, and preferably 20 and more carbon chain length,containing 3 or more double bonds. LCPUFAs of the omega-6 seriesinclude: gamma-linolenic acid (C18:3), di-homo-gammalinolenic acid(C20:3n-6), arachidonic acid (C20:4n-6), adrenic acid (also calleddocosatetraenoic acid or DTA) (C22:4n-6), and docosapentaenoic acid(C22:5n-6). The LCPUFAs of the omega-3 series include: alpha-linolenicacid (C18:3), eicosatrienoic acid (C20:3n-3), eicosatetraenoic acid(C20:4n-3), eicosapentaenoic acid (C20:5n-3), docosapentaenoic acid(C22:5n-3), and docosahexaenoic acid (C22:6n-3). The LCPUFAs alsoinclude fatty acids with greater than 22 carbons and 4 or more doublebonds including but not limited to C28:8(n-3).

Second, a PUFA PKS system according to the present invention comprisesseveral multifunctional proteins (and can include single functionproteins, particularly for PUFA PKS systems from marine bacteria) thatare assembled into a complex that conducts both iterative processing ofthe fatty acid chain as well non-iterative processing, includingtrans-cis isomerization and enoyl reduction reactions in selectedcycles. These proteins can also be referred to herein as the core PUFAPKS enzyme complex or the core PUFA PKS system. The general functions ofthe domains and motifs contained within these proteins are individuallyknown in the art and have been described in detail with regard tovarious PUFA PKS systems from marine bacteria and eukaryotic organisms(see, e.g., U.S. Pat. No. 6,140,486; U.S. Pat. No. 6,566,583; Metz etal., Science 293:290-293 (2001); U.S. Patent Application Publication No.20020194641; U.S. Patent Application Publication No. 20040235127; andU.S. Patent Application Publication No. 20050100995). The domains may befound as a single protein (i.e., the domain and protein are synonymous)or as one of two or more (multiple) domains in a single protein, asmentioned above.

Before the discovery of a PUFA PKS system in marine bacteria (see U.S.Pat. No. 6,140,486), PKS systems were not known to possess thiscombination of iterative and selective enzymatic reactions, and theywere not thought of as being able to produce carbon-carbon double bondsin the cis configuration. However, the PUFA PKS system described by thepresent invention has the capacity to introduce cis double bonds and thecapacity to vary the reaction sequence in the cycle.

The present inventors propose to use these features of the PUFA PKSsystem to produce a range of bioactive molecules that could not beproduced by the previously described (Type I iterative or modular, TypeII, or Type III) PKS systems. These bioactive molecules include, but arenot limited to, polyunsaturated fatty acids (PUFAs), antibiotics orother bioactive compounds, many of which will be discussed below. Forexample, using the knowledge of the PUFA PKS gene structures describedherein, any of a number of methods can be used to alter the PUFA PKSgenes, or combine portions of these genes with other synthesis systems,including other PKS systems, such that new products are produced. Theinherent ability of this particular type of system to do both iterativeand selective reactions will enable this system to yield products thatwould not be found if similar methods were applied to other types of PKSsystems.

Preferably, a PUFA PKS system of the present invention comprises atleast the following biologically active domains that are typicallycontained on three or more proteins: (a) at least one enoyl-ACPreductase (ER) domain; (b) multiple acyl carrier protein (ACP) domain(s)(e.g., at least from one to four, and preferably at least five ACPdomains, and in some embodiments up to six, seven, eight, nine, or morethan nine ACP domains); (c) at least two β-ketoacyl-ACP synthase (KS)domains; (d) at least one acyltransferase (AT) domain; (e) at least oneβ-ketoacyl-ACP reductase (KR) domain; (f) at least two FabA-likeβ-hydroxyacyl-ACP dehydrase (DH) domains; (g) at least one chain lengthfactor (CLF) domain; (h) at least one malonyl-CoA:ACP acyltransferase(MAT) domain. In one embodiment, a PUFA PKS system according to thepresent invention also comprises at least one region containing adehydratase (DH) conserved active site motif.

In a preferred embodiment, a Schizochytrium PUFA PKS system comprises atleast the following biologically active domains: (a) two enoyl-ACPreductase (ER) domain; (b) nine acyl carrier protein (ACP) domains; (c)two β-ketoacyl-ACP synthase (KS) domains; (d) one acyltransferase (AT)domain; (e) one β-ketoacyl-ACP reductase (KR) domain; (f) two FabA-likeβ-hydroxyacyl-ACP dehydrase (DH) domains; (g) one chain length factor(CLF) domain; and (h) one malonyl-CoA:ACP acyltransferase (MAT) domain.In one embodiment, a Schizochytrium PUFA PKS system according to thepresent invention also comprises at least one region or domaincontaining a dehydratase (DH) conserved active site motif that is not apart of a FabA-like DH domain. The structural and functionalcharacteristics of these domains are generally individually known in theart and will be described in detail below with regard to the PUFA PKSsystems of the present invention.

A PUFA PKS system can additionally include one or more accessoryproteins, which are defined herein as proteins that are not consideredto be part of the core PUFA PKS system as described above (i.e., notpart of the PUFA synthase enzyme complex itself), but which may be, orare, necessary for PUFA production or at least for efficient PUFAproduction using the core PUFA synthase enzyme complex of the presentinvention, particularly in certain host organisms (e.g., plants). Forexample, in order to produce PUFAs, a PUFA PKS system must work with anaccessory protein that transfers a 4′-phosphopantetheinyl moiety fromCoenzyme A to the acyl carrier protein (ACP) domain(s). Therefore, aPUFA PKS system can be considered to include at least one4′-phosphopantetheinyl transferase (PPTase) domain, or such a domain canbe considered to be an accessory domain or protein to the PUFA PKSsystem. When genetically modifying organisms (e.g., microorganisms orplants) to express a PUFA PKS system according to the present invention,some host organisms may endogenously express accessory proteins that areneeded to work with the PUFA PKS to produce PUFAs (e.g., PPTases).However, some organisms may be transformed with nucleic acid moleculesencoding one or more accessory proteins described herein to enableand/or to enhance production of PUFAs by the organism, even if theorganism endogenously produces a homologous accessory protein (i.e.,some heterologous accessory proteins may operate more effectively orefficiently with the transformed PUFA synthase proteins than the hostcells' endogenous accessory protein). The present invention provides anexample of bacteria, yeast and plants that have been geneticallymodified with the PUFA PKS system of the present invention that includesan accessory PPTase. Structural and functional characteristics ofPPTases will be described in more detail below.

According to the present invention, reference to a “standard” or“classical” pathway for the production of PUFAs refers to the fatty acidsynthesis pathway where medium chain-length saturated fatty acids(products of a fatty acid synthase (FAS) system) are modified by aseries of elongation and desaturation reactions. The substrates for theelongation reaction are fatty acyl-CoA (the fatty acid chain to beelongated) and malonyl-CoA (the source of the 2 carbons added duringeach elongation reaction). The product of the elongase reaction is afatty acyl-CoA that has two additional carbons in the linear chain. Thedesaturases create cis double bonds in the preexisting fatty acid chainby extraction of 2 hydrogens in an oxygen-dependant reaction. Suchpathways and the genes involved in such pathways are well-known in theliterature.

As used herein, the term “lipid” includes phospholipids (PL); free fattyacids; esters of fatty acids; triacylglycerols (TAG); diacylglycerides;monoacylglycerides; phosphatides; waxes (esters of alcohols and fattyacids); sterols and sterol esters; carotenoids; xanthophylls (e.g.,oxycarotenoids); hydrocarbons; and other lipids known to one of ordinaryskill in the art. The terms “polyunsaturated fatty acid” and “PUFA”include not only the free fatty acid form, but other forms as well, suchas the TAG form and the PL form.

A PUFA PKS system described according to the present invention is anon-bacterial PUFA PKS system. In other words, the PUFA PKS system ofthe present invention is isolated from an organism that is not abacteria, or is a homologue of or derived from a PUFA PKS system from anorganism that is not a bacteria, such as a eukaryote or anarchaebacterium. Eukaryotes are separated from prokaryotes based on thedegree of differentiation of the cells, with eukaryotes being moredifferentiated than prokaryotes. In general, prokaryotes do not possessa nuclear membrane, do not exhibit mitosis during cell division, haveonly one chromosome, their cytoplasm contains 70S ribosomes, they do notpossess any mitochondria, endoplasmic reticulum, chloroplasts, lysosomesor golgi apparatus, their flagella (if present) consists of a singlefibril. In contrast, eukaryotes have a nuclear membrane, they do exhibitmitosis during cell division, they have many chromosomes, theircytoplasm contains 80S ribosomes, they do possess mitochondria,endoplasmic reticulum, chloroplasts (in algae), lysosomes and golgiapparatus, and their flagella (if present) consists of many fibrils. Ingeneral, bacteria are prokaryotes, while algae, fungi, protist, protozoaand higher plants are eukaryotes. The PUFA PKS systems of the marinebacteria (e.g., Shewanella and Vibrio marinus) are not the basis of thepresent invention, although the present invention does contemplate theuse of domains from these bacterial PUFA PKS systems in conjunction withdomains from the non-bacterial (e.g., Schizochytrium) PUFA PKS systemsof the present invention. For example, according to the presentinvention, genetically modified organisms can be produced whichincorporate non-bacterial PUFA PKS functional domains with bacterialPUFA PKS functional domains, as well as PKS functional domains orproteins from other PKS systems (Type I iterative or modular, Type II,or Type III) or FAS systems.

Schizochytrium is a Thraustochytrid marine microorganism thataccumulates large quantities of triacylglycerols rich in DHA anddocosapentaenoic acid (DPA; 22:5%-6); e.g., 30% DHA+DPA by dry weight(Barclay et al., J. Appl. Phycol. 6, 123 (1994)). In eukaryotes thatsynthesize 20- and 22-carbon PUFAs by an elongation/desaturationpathway, the pools of 18-, 20- and 22-carbon intermediates arerelatively large so that in vivo labeling experiments using[¹⁴C]-acetate reveal clear precursor-product kinetics for the predictedintermediates (Gellerman et al., Biochim. Biophys. Acta 573:23 (1979)).Furthermore, radiolabeled intermediates provided exogenously to suchorganisms are converted to the final PUFA products. The presentinventors have shown that [1-¹⁴C]-acetate was rapidly taken up bySchizochytrium cells and incorporated into fatty acids, but at theshortest labeling time (1 min), DHA contained 31% of the label recoveredin fatty acids, and this percentage remained essentially unchangedduring the 10-15 min of [¹⁴C]-acetate acetate incorporation and thesubsequent 24 hours of culture growth (See U.S. Patent ApplicationPublication No. 20020194641, supra). Similarly, DPA represented 10% ofthe label throughout the experiment. There is no evidence for aprecursor-product relationship between 16- or 18-carbon fatty acids andthe 22-carbon polyunsaturated fatty acids. These results are consistentwith rapid synthesis of DHA from [¹⁴C]-acetate involving very small(possibly enzyme-bound) pools of intermediates. A cell-free homogenatederived from Schizochytrium cultures incorporated [1-¹⁴C]-malonyl-CoAinto DHA, DPA, and saturated fatty acids. The same biosyntheticactivities were retained by a 100,000×g supernatant fraction but werenot present in the membrane pellet. Thus, DHA and DPA synthesis inSchizochytrium does not involve membrane-bound desaturases or fatty acidelongation enzymes like those described for other eukaryotes(Parker-Barnes et al., 2000, supra; Shanklin et al., 1998, supra). Thesefractionation data contrast with those obtained from the Shewanellaenzymes (See Metz et al., 2001, supra) and may indicate use of adifferent (soluble) acyl acceptor molecule, such as CoA, by theSchizochytrium enzyme.

As described in U.S. Pat. No. 6,566,583, a cDNA library fromSchizochytrium was constructed and approximately 8,000 random clones(ESTs) were sequenced. Within this dataset, only one moderatelyexpressed gene (0.3% of all sequences) was identified as a fatty aciddesaturase, although a second putative desaturase was represented by asingle clone (0.01%). By contrast, sequences that exhibited homology to8 of the 11 domains of the Shewanella PKS genes shown in FIG. 2 were allidentified at frequencies of 0.2-0.5%. In U.S. Pat. No. 6,566,583,several cDNA clones showing homology to the Shewanella PKS genes weresequenced, and various clones were assembled into nucleic acid sequencesrepresenting two partial open reading frames and one complete openreading frame.

Nucleotides 390-4443 of the cDNA sequence containing the first partialopen reading frame described in U.S. application Ser. No. 09/231,899(denoted therein as SEQ ID NO:69) match nucleotides 4677-8730 (plus thestop codon) of the sequence denoted herein as OrfA (SEQ ID NO:1). A cDNAclone described in U.S. application Ser. No. 09/231,899 as cDNA cloneLIB3033-047-B5 comprises at least a portion of nucleotides 4677-8730 ofSEQ ID NO:1 described herein, to the best of the present inventors'knowledge. Specifically, the sequence of the insert in cDNA cloneLIB3033-047-B5 begins at nucleotide 6719 of SEQ ID NO:1 and extends tothe end of the Orf (position 8730 of SEQ ID NO:1), plus about 71nucleotides beyond the end of the Orf represented by SEQ ID NO:1. cDNAclone LIB3033-047-B5 (denoted cDNA clone LIB3033-047-B5 in the form ofan E. coli plasmid vector containing “Orf6 homolog” partial genesequence from Schizochytrium sp.) was deposited with the American TypeCulture Collection (ATCC), University Boulevard, Manassas, Va.20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______.The nucleotide sequence of cDNA clone LIB3033-047-B5, and the amino acidsequence encoded by this cDNA clone are encompassed by the presentinvention. A second cDNA clone described in U.S. application Ser. No.09/231,899 as cDNA clone LIB3033-046-E6 shared homology to the ACPdomain of ORF6, contained 6 ACP repeats, and comprises at least aportion of nucleotides of SEQ ID NO:1 of the present invention. ThiscDNA clone did not have a poly-A-tail, and therefore, was a partial cDNAwith additional regions of the cDNA found downstream of the sequence.The nucleotide sequence of cDNA clone LIB3033-046-E6, and the amino acidsequence encoded by this cDNA clone are encompassed by the presentinvention.

Nucleotides 1-4867 of the cDNA sequence containing the second partialopen reading frame described in U.S. application Ser. No. 09/231,899(denoted therein as SEQ ID NO:71) matches nucleotides 1311-6177 (plusthe stop codon) of the sequence denoted herein as OrfB (SEQ ID NO:3),with the exception of the nucleotide at position 2933 of SEQ ID NO:71 ofthe '899 application, which corresponds to the nucleotide at position4243 of SEQ ID NO:3 set forth herein. This single nucleotide change (Cto G) results in a single amino acid change in SEQ ID NO:4 disclosedherein, as compared to SEQ ID NO:72 of the '899 application.Specifically, the glutamine residue at position 978 of SEQ ID NO:72 inthe '899 application is changed to a glutamate at position 1415 of SEQID NO:4. This amino acid occurs in the linker region between the ATdomain and the ER domain of SEQ ID NO:4. A cDNA clone described in U.S.application Ser. No. 09/231,899 as cDNA clone LIB3033-046-D2 comprisesnucleotides 1311-6177 of SEQ ID NO:3 described herein, plus about 382additional nucleotides beyond the end of the Orf represented here as SEQID NO:3, to the best of the present inventors' knowledge. cDNA cloneLIB3033-046-D2 (denoted cDNA clone LIB3033-046-D2 in the form of an E.coli plasmid vector containing “hglC/Orf7/Orf8/Orf9 homolog” gene fromSchizochytrium) was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun.8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequenceof cDNA clone LIB3033-046-D2, and the amino acid sequence encoded bythis cDNA clone are encompassed by the present invention.

Nucleotides 145-4653 of the cDNA sequence containing the complete openreading frame described in U.S. application Ser. No. 09/231,899 (denotedtherein as SEQ ID NO:76 and incorrectly designated as a partial openreading frame) matches nucleotides 1-2624 and 2675-4506 of the sequencedenoted herein as OrfC (SEQ ID NO:5). Sequencing of the genomic DNAencoding OrfC revealed that there is an additional nucleotide at each ofpositions 2769, 2806 and 2818 of SEQ ID NO:76 of the '899 applicationwhich resulted in a frame shift and a short change in the amino acidsequence of the corresponding protein. Therefore, amino acid positions924-939 of SEQ ID NO:73 of the '899 application represent an incorrectsequence. Positions 876-890 of SEQ ID NO:5 herein represent the correctamino acid sequence in this region. This sequence is located in the DH2domain of OrfC (discussed below). A cDNA clone described in U.S.application Ser. No. 09/231,899 as cDNA clone LIB81-042-B9 comprises aportion of the 5′ sequence of SEQ ID NO:5. To the best of the presentinventors' knowledge, the sequence of the insert in LIB81-042-B9contains 145 nucleotides upstream of the start codon of SEQ ID NO:5 andextends 2361 nucleotides into the Orf. cDNA clone LIB81-042-B9 (denotedcDNA clone LIB81-042-B9 in the form of an E. coli plasmid vectorcontaining “0118 homolog” partial gene sequence from Schizochytrium sp.)was deposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, andassigned ATCC Accession No. ______. The nucleotide sequence of cDNAclone LIB81-042-B9, and the amino acid sequence encoded by this cDNAclone are encompassed by the present invention. A second cDNA clonedescribed in U.S. application Ser. No. 09/231,899 as cDNA cloneLIB81-015-D5 aligns with Shewanella ORF8 and also with Shewanella ORF9.The open reading frame of LIB81-015-D5 aligns with SEQ ID NO:5 beginningat nucleotide 2526 of SEQ ID NO:5 and extends to the end of the Orf(i.e., position 4506), plus about 115 bp including a poly A tail beyondSEQ ID NO:5. The nucleotide sequence of cDNA clone LIB81-015-D5, and theamino acid sequence encoded by this cDNA clone are encompassed by thepresent invention.

Further sequencing of cDNA and genomic clones by the present inventorsallowed the identification of the full-length genomic sequence of eachof OrfA, OrfB and OrfC in Schizochytrium, including in Schizochytriumsp. ATCC 20888 and the mutated daughter strain of ATCC 20888, denotedSchizochytrium sp., strain N230D. N230D was one of more than 1,000randomly-chosen survivors of chemically mutagenised (NTG;1-methyl-3-nitro-1-nitrosoguanidine) Schizochytrium ATCC 20888 screenedfor variations in fatty acid content. This particular strain was valuedfor its improved DHA productivity. Further, the complete identificationof the domains with homology to those in Shewanella (see FIG. 2) wereidentified. It is noted that in Schizochytrium, the Orfs of the genomicDNA and cDNA are identical, due to the lack of introns in the organismgenome, to the best of the present inventors' knowledge. Therefore,reference to a nucleotide sequence of Orfs from Schizochytrium can referto genomic DNA or cDNA.

FIG. 1 is a graphical representation of the three open reading framesfrom the Schizochytrium PUFA PKS system, and includes the domainstructure of this PUFA PKS system. The domain structure of each openreading frame is as follows:

Open Reading Frame A (OrfA):

The complete nucleotide sequence for OrfA is represented herein as SEQID NO:1. Nucleotides 4677-8730 of SEQ ID NO:1 correspond to nucleotides390-4443 of the sequence denoted as SEQ ID NO:69 in U.S. applicationSer. No. 09/231,899. Therefore, nucleotides 1-4676 of SEQ ID NO:1represent additional sequence that was not disclosed in U.S. applicationSer. No. 09/231,899. This novel region of SEQ ID NO:1 encodes thefollowing domains in OrfA: (1) the ORFA-KS domain; (2) the ORFA-MATdomain; and (3) at least a portion of the ACP domain region (e.g., atleast ACP domains 1-4). It is noted that nucleotides 1-389 of SEQ IDNO:69 in U.S. application Ser. No. 09/231,899 do not exactly match withthe 389 nucleotides that are upstream of position 4677 in SEQ ID NO:1disclosed herein. Therefore, positions 1-389 of SEQ ID NO:69 in U.S.application Ser. No. 09/231,899 appear to be incorrectly placed next tonucleotides 390-4443 of that sequence. Most of these first 389nucleotides (about positions 60-389) are a match with an upstreamportion of OrfA (SEQ ID NO:1) of the present invention and therefore, itis believed that an error occurred in the effort to prepare the contigof the cDNA constructs in U.S. application Ser. No. 09/231,899. Theregion in which the alignment error occurred in U.S. application Ser.No. 09/231,899 is within the region of highly repetitive sequence (i.e.,the ACP region, discussed below), which probably created some confusionin the assembly of that sequence from various cDNA clones.

OrfA is a 8730 nucleotide sequence (not including the stop codon) whichencodes a 2910 amino acid sequence, represented herein as SEQ ID NO:2.Within OrfA are twelve domains: (a) one β-keto acyl-ACP synthase (KS)domain; (b) one malonyl-CoA:ACP acyltransferase (MAT) domain; (c) nineacyl carrier protein (ACP) domains; and (d) one ketoreductase (KR)domain.

A nucleotide sequence for OrfA has been deposited with GenBank asAccession No. AF378327 (amino acid sequence Accession No. AAK728879).The nucleotide sequence represented by GenBank Accession No. AF378327differs from the sequence represented herein as SEQ ID NO:1 by the pointnucleotide changes: (1) at position 1999 (A to G, resulting in an aminoacid change from an asparagine to an aspartic acid at position 667 ofSEQ ID NO:2); (2) at position 2003 (C to A, resulting in an amino acidchange from a proline to a histidine at position 668 of SEQ ID NO:2);and (3) at position 2238 (A to C, resulting in no amino acid change atposition 746 of SEQ ID NO:2). Each of the two amino acid changes fromthe amino acid sequence encoded by GenBank Accession No. AAK728879 arelocated in the MAT domain (SEQ ID NO:10) of SEQ ID NO:2.

Genomic DNA clones (plasmids) encoding OrfA from both Schizochytrium sp.ATCC 20888 and a daughter strain of ATCC 20888, denoted Schizochytriumsp., strain N230D, have been isolated and sequenced. A genomic clonedescribed herein as JK1126, isolated from Schizochytrium sp. ATCC 20888,comprises, to the best of the present inventors' knowledge, thenucleotide sequence spanning from position 1 to 4119 and from position5498 to 8730 of SEQ ID NO:1, and encodes the corresponding amino acidsequence of SEQ ID NO:2. Indeed, it is expected that JK1126 comprisesSEQ ID NO:1 in its entirety and encodes SEQ ID NO:2. Genomic clonepJK1126 (denoted pJK1126 OrfA genomic clone, in the form of an E. coliplasmid vector containing “OrfA” gene from Schizochytrium ATCC 20888)was deposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, andassigned ATCC Accession No. ______. The nucleotide sequence of pJK1126OrfA genomic clone, and the amino acid sequence encoded by this plasmidare encompassed by the present invention.

Two genomic clones described herein as pJK306 OrfA genomic clone andpJK320 OrfA genomic clone, isolated from Schizochytrium sp. N230D,together (overlapping clones) comprise, to the best of the presentinventors' knowledge, the nucleotide sequence of SEQ ID NO:1, and encodethe amino acid sequence of SEQ ID NO:2. Genomic clone pJK306 (denotedpJK306 OrfA genomic clone, in the form of an E. coli plasmid containing5′ portion of OrfA gene from Schizochytrium sp. N230D (2.2 kB overlapwith pJK320)) was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun.8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequenceof pJK306 OrfA genomic clone, and the amino acid sequence encoded bythis plasmid are encompassed by the present invention. Genomic clonepJK320 (denoted pJK320 OrfA genomic clone, in the form of an E. coliplasmid containing 3′ portion of OrfA gene from Schizochytrium sp. N230D(2.2 kB overlap with pJK306)) was deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______.The nucleotide sequence of pJK320 OrfA genomic clone, and the amino acidsequence encoded by this plasmid are encompassed by the presentinvention.

OrfA was compared with known sequences in a standard BLAST search (BLAST2.0 Basic BLAST homology search using blastp for amino acid searches,blastn for nucleic acid searches, and blastX for nucleic acid searchesand searches of the translated amino acid sequence in all 6 open readingframes with standard default parameters, wherein the query sequence isfiltered for low complexity regions by default (described in Altschul,S.F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W.& Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs.” Nucleic Acids Res. 25:3389-3402,incorporated herein by reference in its entirety)). At the nucleic acidlevel, OrfA has no significant homology to any known nucleotidesequence. At the amino acid level, the sequences with the greatestdegree of homology to ORFA were: Nostoc sp. 7120 heterocyst glycolipidsynthase (Accession No. NC_(—)003272), which was 42% identical to ORFAover 1001 amino acid residues; and Moritella marinas (Vibrio marinas)ORF8 (Accession No. AB025342), which was 40% identical to ORFA over 993amino acid residues.

The first domain in OrfA is a KS domain, also referred to herein asORFA-KS. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 40 ofSEQ ID NO:1 (OrfA) to an ending point of between about positions 1428and 1500 of SEQ ID NO:1 (based on homology to other PUFA PKS domains,the position of the domain spans from about position 1 to about position1500; based on Pfam analysis, a KS core region spans from about position40 to about position 1428). The nucleotide sequence containing thesequence encoding the ORFA-KS domain is represented herein as SEQ IDNO:7 (positions 1-1500 of SEQ ID NO:1). The amino acid sequencecontaining the KS domain spans from a starting point of between aboutpositions 1 and 14 of SEQ ID NO:2 (ORFA) to an ending point of betweenabout positions 476 and 500 of SEQ ID NO:2 (again, referring to theoverall homology to PUFA PKS KS domains and to Pfam core regions,respectively). The amino acid sequence containing the ORFA-KS domain isrepresented herein as SEQ ID NO:8 (positions 1-500 of SEQ ID NO:2). Itis noted that the ORFA-KS domain contains an active site motif: DXAC*(*acyl binding site C₂₁₅).

According to the present invention, a domain or protein having 3-ketoacyl-ACP synthase (KS) biological activity (function) is characterizedas the enzyme that carries out the initial step of the FAS (and PKS)elongation reaction cycle. The acyl group destined for elongation islinked to a cysteine residue at the active site of the enzyme by athioester bond. In the multi-step reaction, the acyl-enzyme undergoescondensation with malonyl-ACP to form -keto acyl-ACP, CO₂ and freeenzyme. The KS plays a key role in the elongation cycle and in manysystems has been shown to possess greater substrate specificity thanother enzymes of the reaction cycle. For example, E. coli has threedistinct KS enzymes—each with its own particular role in the physiologyof the organism (Magnuson et al., Microbiol. Rev. 57, 522 (1993)). Thetwo KS domains of the PUFA-PKS systems could have distinct roles in thePUFA biosynthetic reaction sequence.

As a class of enzymes, KS's have been well characterized. The sequencesof many verified KS genes are know, the active site motifs have beenidentified and the crystal structures of several have been determined.Proteins (or domains of proteins) can be readily identified as belongingto the KS family of enzymes by homology to known KS sequences.

The second domain in OrfA is a MAT domain, also referred to herein asORFA-MAT. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1723 and 1798of SEQ ID NO:1 (OrfA) to an ending point of between about positions 2805and 3000 of SEQ ID NO:1 (based on homology to other PUFA PKS domains,the position of the MAT domain spans from about position 1723 to aboutposition 3000; based on Pfam analysis, a MAT core region spans fromabout position 1798 to about position 2805). The nucleotide sequencecontaining the sequence encoding the ORFA-MAT domain is representedherein as SEQ ID NO:9 (positions 1723-3000 of SEQ ID NO:1). The aminoacid sequence containing the MAT domain spans from a starting point ofbetween about positions 575 and 600 of SEQ ID NO:2 (ORFA) to an endingpoint of between about positions 935 and 1000 of SEQ ID NO:2 (again,referring to the overall homology to PUFA PKS MAT domains and to Pfamcore regions, respectively). The amino acid sequence containing theORFA-MAT domain is represented herein as SEQ ID NO:10 (positions575-1000 of SEQ ID NO:2). The MAT domain comprises an aspartate atposition 93 and a histidine at position 94 (corresponding to positions667 and 668, respectively, of SEQ ID NO:2). It is noted that theORFA-MAT domain contains an active site motif: GHS*XG (*acyl bindingsite S₇₀₆), represented herein as SEQ ID NO:11.

According to the present invention, a domain or protein havingmalonyl-CoA:ACP acyltransferase (MAT) biological activity (function) ischaracterized as one that transfers the malonyl moiety from malonyl-CoAto ACP. In addition to the active site motif (GxSxG), these enzymespossess an extended motif R and Q amino acids in key positions) thatidentifies them as MAT enzymes (in contrast to the AT domain ofSchizochytriumOrf B). In some PKS systems (but not the PUFA PKS domain)MAT domains will preferentially load methyl- or ethyl-malonate on to theACP group (from the corresponding CoA ester), thereby introducingbranches into the linear carbon chain. MAT domains can be recognized bytheir homology to known MAT sequences and by their extended motifstructure.

Domains 3-11 of OrfA are nine tandem ACP domains, also referred toherein as ORFA-ACP (the first domain in the sequence is ORFA-ACP1, thesecond domain is ORFA-ACP2, the third domain is ORFA-ACP3, etc.). Thefirst ACP domain, ORFA-ACP1, is contained within the nucleotide sequencespanning from about position 3343 to about position 3600 of SEQ ID NO:1(OrfA). The nucleotide sequence containing the sequence encoding theORFA-ACP1 domain is represented herein as SEQ ID NO:12 (positions3343-3600 of SEQ ID NO:1). The amino acid sequence containing the firstACP domain spans from about position 1115 to about position 1200 of SEQID NO:2. The amino acid sequence containing the ORFA-ACP1 domain isrepresented herein as SEQ ID NO:13 (positions 1115-1200 of SEQ ID NO:2).It is noted that the ORFA-ACP1 domain contains an active site motif:LGIDS* (*pantetheine binding motif S₁₁₅₇), represented herein by SEQ IDNO:14.

The nucleotide and amino acid sequences of all nine ACP domains arehighly conserved and therefore, the sequence for each domain is notrepresented herein by an individual sequence identifier. However, basedon the information disclosed herein, one of skill in the art can readilydetermine the sequence containing each of the other eight ACP domains(see discussion below).

All nine ACP domains together span a region of OrfA of from aboutposition 3283 to about position 6288 of SEQ ID NO:1, which correspondsto amino acid positions of from about 1095 to about 2096 of SEQ ID NO:2.The nucleotide sequence for the entire ACP region containing all ninedomains is represented herein as SEQ ID NO:16. The region represented bySEQ ID NO:16 includes the linker segments between individual ACPdomains. The repeat interval for the nine domains is approximately every330 nucleotides of SEQ ID NO:16 (the actual number of amino acidsmeasured between adjacent active site serines ranges from 104 to 116amino acids). Each of the nine ACP domains contains a pantetheinebinding motif LGIDS* (represented herein by SEQ ID NO:14), wherein S* isthe pantetheine binding site serine (S). The pantetheine binding siteserine (S) is located near the center of each ACP domain sequence. Ateach end of the ACP domain region and between each ACP domain is aregion that is highly enriched for proline (P) and alanine (A), which isbelieved to be a linker region. For example, between ACP domains 1 and 2is the sequence: APAPVKAAAPAAPVASAPAPA, represented herein as SEQ IDNO:15. The locations of the active site serine residues (i.e., thepantetheine binding site) for each of the nine ACP domains, with respectto the amino acid sequence of SEQ ID NO:2, are as follows: ACP1=S₁₁₅₇;ACP2=S₁₂₆₆; ACP3=S₁₃₇₇; ACP4=S₁₄₈₈; ACP5=S₁₆₀₄; ACP6=S₁₇₁₅; ACP7=S₁₈₁₉;ACP8=S₁₉₃₀; and ACP9=S₂₀₃₄. Given that the average size of an ACP domainis about 85 amino acids, excluding the linker, and about 110 amino acidsincluding the linker, with the active site serine being approximately inthe center of the domain, one of skill in the art can readily determinethe positions of each of the nine ACP domains in OrfA.

According to the present invention, a domain or protein having acylcarrier protein (ACP) biological activity (function) is characterized asbeing small polypeptides (typically, 80 to 100 amino acids long), thatfunction as carriers for growing fatty acyl chains via a thioesterlinkage to a covalently bound co-factor of the protein. They occur asseparate units or as domains within larger proteins. ACPs are convertedfrom inactive apo-forms to functional holo-forms by transfer of thephosphopantetheinyl moiety of CoA to a highly conserved serine residueof the ACP. Acyl groups are attached to ACP by a thioester linkage atthe free terminus of the phosphopantetheinyl moiety. ACPs can beidentified by labeling with radioactive pantetheine and by sequencehomology to known ACPs. The presence of variations of the abovementioned motif (LGIDS*) is also a signature of an ACP.

Domain 12 in OrfA is a KR domain, also referred to herein as ORFA-KR.This domain is contained within the nucleotide sequence spanning from astarting point of about position 6598 of SEQ ID NO:1 to an ending pointof about position 8730 of SEQ ID NO:1. The nucleotide sequencecontaining the sequence encoding the ORFA-KR domain is representedherein as SEQ ID NO:17 (positions 6598-8730 of SEQ ID NO:1). The aminoacid sequence containing the KR domain spans from a starting point ofabout position 2200 of SEQ ID NO:2 (ORFA) to an ending point of aboutposition 2910 of SEQ ID NO:2. The amino acid sequence containing theORFA-KR domain is represented herein as SEQ ID NO:18 (positions2200-2910 of SEQ ID NO:2). Within the KR domain is a core region withhomology to short chain aldehyde-dehydrogenases (KR is a member of thisfamily). This core region spans from about position 7198 to aboutposition 7500 of SEQ ID NO:1, which corresponds to amino acid positions2400-2500 of SEQ ID NO:2.

According to the present invention, a domain or protein havingketoreductase activity, also referred to as 3-ketoacyl-ACP reductase(KR) biological activity (function), is characterized as one thatcatalyzes the pyridine-nucleotide-dependent reduction of 3-keto acylforms of ACP. It is the first reductive step in the de novo fatty acidbiosynthesis elongation cycle and a reaction often performed inpolyketide biosynthesis. Significant sequence similarity is observedwith one family of enoyl ACP reductases (ER), the other reductase of FAS(but not the ER family present in the PUFA PKS system), and theshort-chain alcohol dehydrogenase family. Pfam analysis of the PUFA PKSregion indicated above reveals the homology to the short-chain alcoholdehydrogenase family in the core region. Blast analysis of the sameregion reveals matches in the core area to known KR enzymes as well asan extended region of homology to domains from the other characterizedPUFA PKS systems.

Open Reading Frame B (OrfB):

The complete nucleotide sequence for OrfB is represented herein as SEQID NO:3. Nucleotides 1311-6177 of SEQ ID NO:3 correspond to nucleotides1-4867 of the sequence denoted as SEQ ID NO:71 in U.S. application Ser.No. 09/231,899, with the exception of the nucleotide at position 2933 ofSEQ ID NO:71 of the '899 application or nucleotide 4243 of SEQ ID NO:3herein, as discussed above. The cDNA sequence in U.S. application Ser.No. 09/231,899 contains about 345 additional nucleotides beyond the stopcodon, including a polyA tail). Therefore, nucleotides 1-1310 of SEQ IDNO:1 represent additional sequence that was not disclosed in U.S.application Ser. No. 09/231,899. This novel region of SEQ ID NO:3contains most of the KS domain encoded by OrfB.

OrfB is a 6177 nucleotide sequence (not including the stop codon) whichencodes a 2059 amino acid sequence, represented herein as SEQ ID NO:4.Within OrfB are four domains: (a) one β-keto acyl-ACP synthase (KS)domain; (b) one chain length factor (CLF) domain; (c) one acyltransferase (AT) domain; and, (d) one enoyl ACP-reductase (ER) domain.

A nucleotide sequence for OrfB has been deposited with GenBank asAccession No. AF378328 (amino acid sequence Accession No. AAK728880).The nucleotide sequence represented by GenBank Accession No. AF378328differs from the nucleotide sequence represented herein as SEQ ID NO:3by the point nucleotide changes: (1) at position 852 (T to C, resultingin no amino acid change at position 284 of SEQ ID NO:4); (2) at position1110 (S to C, resulting in no amino acid change at position 370 of SEQID NO:4); (3) at position 1112 (Y to T, resulting in the resolution ofan ambiguous amino acid call to a definite valine call at position 371of SEQ ID NO:4); and (4) at position 4243 (C to G, resulting in a changefrom a glutamine to a glutamate at position 1415 of SEQ ID NO:4). Thesingle amino acid change from the amino acid sequence encoded by GenBankAccession No. AAK728880 is located in the linker region located betweenthe AT domain and the ER domain of SEQ ID NO:4.

Genomic DNA clones (plasmids) encoding OrfB from both Schizochytrium sp.ATCC 20888 and a daughter strain of ATCC 20888, denoted Schizochytriumsp., strain N230D, have been isolated and sequenced. A genomic clonedescribed herein as pJK1129, isolated from Schizochytrium sp. ATCC20888, comprises, to the best of the present inventors' knowledge, thenucleotide sequence of SEQ ID NO:3, and encodes the amino acid sequenceof SEQ ID NO:4. Genomic clone pJK1129 (denoted pJK1129 OrfB genomicclone, in the form of an E. coli plasmid vector containing “OrfB” genefrom Schizochytrium ATCC 20888) was deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______.The nucleotide sequence of pJK1126 OrfB genomic clone, and the aminoacid sequence encoded by this plasmid are encompassed by the presentinvention.

A genomic clone described herein as pJK324 OrfB genomic clone, isolatedfrom Schizochytrium sp. N230D, comprises, to the best of the presentinventors' knowledge, the nucleotide sequence of SEQ ID NO:3, andencodes the amino acid sequence of SEQ ID NO:4. Genomic clone pJK324(denoted pJK324 OrfB genomic clone, in the form of an E. coli plasmidcontaining the OrfB gene sequence from Schizochytrium sp. N230D) wasdeposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, andassigned ATCC Accession No. ______. The nucleotide sequence of pJK324OrfB genomic clone, and the amino acid sequence encoded by this plasmidare encompassed by the present invention.

OrfB was compared with known sequences in a standard BLAST search asdescribed above. At the nucleic acid level, OrfB has no significanthomology to any known nucleotide sequence. At the amino acid level, thesequences with the greatest degree of homology to ORFB were: Shewanellasp. hypothetical protein (Accession No. U73935), which was 53% identicalto ORFB over 458 amino acid residues; Moritella marinus (Vibrio marinus)ORF11 (Accession No. AB025342), which was 53% identical to ORFB over 460amino acid residues; Photobacterium profundum omega-3 polyunsaturatedfatty acid synthase PfaD (Accession No. AF409100), which was 52%identical to ORFB over 457 amino acid residues; and Nostoc sp. 7120hypothetical protein (Accession No. NC_(—)003272), which was 53%identical to ORFB over 430 amino acid residues.

The first domain in OrfB is a KS domain, also referred to herein asORFB-KS. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 43 ofSEQ ID NO:3 (OrfB) to an ending point of between about positions 1332and 1350 of SEQ ID NO:3 (based on homology to other PUFA PKS domains,the position of the KS domain spans from about position 1 to aboutposition 1350; based on Pfam analysis, a KS core region spans from aboutposition 43 to about position 1332). The nucleotide sequence containingthe sequence encoding the ORFB-KS domain is represented herein as SEQ IDNO:19 (positions 1-1350 of SEQ ID NO:3). The amino acid sequencecontaining the KS domain spans from a starting point of between aboutpositions 1 and 15 of SEQ ID NO:4 (ORFB) to an ending point of betweenabout positions 444 and 450 of SEQ ID NO:4 (again, referring to theoverall homology to PUFA PKS KS domains and to Pfam core regions,respectively). The amino acid sequence containing the ORFB-KS domain isrepresented herein as SEQ ID NO:20 (positions 1-450 of SEQ ID NO:4).This KS domain comprises a valine at position 371 of SEQ ID NO:20 (alsoposition 371 of SEQ ID NO:20). It is noted that the ORFB-KS domaincontains an active site motif: DXAC* (*acyl binding site C₁₉₆). KSbiological activity and methods of identifying proteins or domainshaving such activity is described above.

The second domain in OrfB is a CLF domain, also referred to herein asORFB-CLF. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1378 and 1402of SEQ ID NO:3 (OrfB) to an ending point of between about positions 2682and 2700 of SEQ ID NO:3 (based on homology to other PUFA PKS domains,the position of the CLF domain spans from about position 1378 to aboutposition 2700; based on Pfam analysis, a CLF core region spans fromabout position 1402 to about position 2682). The nucleotide sequencecontaining the sequence encoding the ORFB-CLF domain is representedherein as SEQ ID NO:21 (positions 1378-2700 of SEQ ID NO:3). The aminoacid sequence containing the CLF domain spans from a starting point ofbetween about positions 460 and 468 of SEQ ID NO:4 (ORFB) to an endingpoint of between about positions 894 and 900 of SEQ ID NO:4 (again,referring to the overall homology to PUFA PKS CLF domains and to Pfamcore regions, respectively). The amino acid sequence containing theORFB-CLF domain is represented herein as SEQ ID NO:22 (positions 460-900of SEQ ID NO:4). It is noted that the ORFB-CLF domain contains a KSactive site motif without the acyl-binding cysteine.

According to the present invention, a domain or protein is referred toas a chain length factor (CLF) based on the following rationale. The CLFwas originally described as characteristic of Type II (dissociatedenzymes) PKS systems and was hypothesized to play a role in determiningthe number of elongation cycles, and hence the chain length, of the endproduct. CLF amino acid sequences show homology to KS domains (and arethought to form heterodimers with a KS protein), but they lack theactive site cysteine. CLF's role in PKS systems is currentlycontroversial. New evidence (C. Bisang et al., Nature 401, 502 (1999))suggests a role in priming (providing the initial acyl group to beelongated) the PKS systems. In this role the CLF domain is thought todecarboxylate malonate (as malonyl-ACP), thus forming an acetate groupthat can be transferred to the KS active site. This acetate thereforeacts as the ‘priming’ molecule that can undergo the initial elongation(condensation) reaction. Homologues of the Type II CLF have beenidentified as ‘loading’ domains in some modular PKS systems. A domainwith the sequence features of the CLF is found in all currentlyidentified PUFA PKS systems and in each case is found as part of amultidomain protein.

The third domain in OrfB is an AT domain, also referred to herein asORFB-AT. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 2701 and 3598of SEQ ID NO:3 (OrfB) to an ending point of between about positions 3975and 4200 of SEQ ID NO:3 (based on homology to other PUFA PKS domains,the position of the AT domain spans from about position 2701 to aboutposition 4200; based on Pfam analysis, an AT core region spans fromabout position 3598 to about position 3975). The nucleotide sequencecontaining the sequence encoding the ORFB-AT domain is representedherein as SEQ ID NO:23 (positions 2701-4200 of SEQ ID NO:3). The aminoacid sequence containing the AT domain spans from a starting point ofbetween about positions 901 and 1200 of SEQ ID NO:4 (ORFB) to an endingpoint of between about positions 1325 and 1400 of SEQ ID NO:4 (again,referring to the overall homology to PUFA PKS AT domains and to Pfamcore regions, respectively). The amino acid sequence containing theORFB-AT domain is represented herein as SEQ ID NO:24 (positions 901-1400of SEQ ID NO:4). It is noted that the ORFB-AT domain contains an activesite motif of GxS*xG (*acyl binding site S₁₁₄₀) that is characteristicof acyltransferse (AT) proteins.

An “acyltransferase” or “AT” refers to a general class of enzymes thatcan carry out a number of distinct acyl transfer reactions. TheSchizochytrium domain shows good homology to a domain present in all ofthe other PUFA PKS systems currently examined and very weak homology tosome acyltransferases whose specific functions have been identified(e.g. to malonyl-CoA:ACP acyltransferase, MAT). In spite of the weakhomology to MAT, this AT domain is not believed to function as a MATbecause it does not possess an extended motif structure characteristicof such enzymes (see MAT domain description, above). For the purposes ofthis disclosure, the functions of the AT domain in a PUFA PKS systeminclude, but are not limited to: transfer of the fatty acyl group fromthe ORFA ACP domain(s) to water (i.e. a thioesterase—releasing the fattyacyl group as a free fatty acid), transfer of a fatty acyl group to anacceptor such as CoA, transfer of the acyl group among the various ACPdomains, or transfer of the fatty acyl group to a lipophilic acceptormolecule (e.g. to lysophosphadic acid).

The fourth domain in OrfB is an ER domain, also referred to herein asORFB-ER. This domain is contained within the nucleotide sequencespanning from a starting point of about position 4648 of SEQ ID NO:3(OrfB) to an ending point of about position 6177 of SEQ ID NO:3. Thenucleotide sequence containing the sequence encoding the ORFB-ER domainis represented herein as SEQ ID NO:25 (positions 4648-6177 of SEQ IDNO:3). The amino acid sequence containing the ER domain spans from astarting point of about position 1550 of SEQ ID NO:4 (ORFB) to an endingpoint of about position 2059 of SEQ ID NO:4. The amino acid sequencecontaining the ORFB-ER domain is represented herein as SEQ ID NO:26(positions 1550-2059 of SEQ ID NO:4).

According to the present invention, this domain has enoyl reductase (ER)biological activity. The ER enzyme reduces the trans-double bond(introduced by the DH activity) in the fatty acyl-ACP, resulting infully saturating those carbons. The ER domain in the PUFA-PKS showshomology to a newly characterized family of ER enzymes (Heath et al.,Nature 406, 145 (2000)). Heath and Rock identified this new class of ERenzymes by cloning a gene of interest from Streptococcus pneumoniae,purifying a protein expressed from that gene, and showing that it had ERactivity in an in vitro assay. The sequence of the Schizochytrium ERdomain of OrfB shows homology to the S. pneumoniae ER protein. All ofthe PUFA PKS systems currently examined contain at least one domain withvery high sequence homology to the Schizochytrium ER domain. TheSchizochytrium PUFA PKS system contains two ER domains (one on OrfB andone on OrfC).

Open Reading Frame C (OrfC):

The complete nucleotide sequence for OrfC is represented herein as SEQID NO:5. Nucleotides 1-4506 of SEQ ID NO:5 (i.e., the entire openreading frame sequence, not including the stop codon) nearly correspondto nucleotides 145-4653 of the sequence denoted as SEQ ID NO:76 in U.S.application Ser. No. 09/231,899. The cDNA sequence in U.S. applicationSer. No. 09/231,899 contains about 144 nucleotides upstream of the startcodon for OrfC and about 110 nucleotides beyond the stop codon,including a polyA tail. In addition, as discussed above, nucleotides145-4653 of the cDNA sequence containing the complete open reading framedescribed in U.S. application Ser. No. 09/231,899 (denoted therein asSEQ ID NO:76) match nucleotides 1-2624 and 2675-4506 of the sequencedenoted herein as OrfC (SEQ ID NO:5). OrfC is a 4506 nucleotide sequence(not including the stop codon) which encodes a 1502 amino acid sequence,represented herein as SEQ ID NO:6. Within OrfC are three domains: (a)two FabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; and (b) oneenoyl ACP-reductase (ER) domain.

A nucleotide sequence for OrfC has been deposited with GenBank asAccession No. AF378329 (amino acid sequence Accession No. AAK728881).The nucleotide sequence represented by AF378329 differs from thenucleotide sequence represented herein as SEQ ID NO:5 by the pointnucleotide insertions: (1) at position 2625 (an insertion of an A); (2)at position 2662 (an insertion of a C); and (3) at position 2674 (aninsertion of an A). This resulted in a frame shift out of frame atposition 2625 and then back into frame at position 2675. The amino acidsequence encoded by GenBank Accession No. AAK728881 differs from theamino acid sequence encoded by SEQ ID NO:5 (i.e., SEQ ID NO:6) in theregion spanning from positions 876-891 of GenBank Accession No.AAK728881 or positions 876-890 of SEQ ID NO:6. This change in sequenceoccurs in the DH2 domain of OrfC (discussed below).

Genomic DNA clones (plasmids) encoding OrfC from both Schizochytrium sp.ATCC 20888 and a daughter strain of ATCC 20888, denoted Schizochytriumsp., strain N230D, have been isolated and sequenced. A genomic clonedescribed herein as pJK1131, isolated from Schizochytrium sp. ATCC20888, comprises, to the best of the present inventors' knowledge, thenucleotide sequence of SEQ ID NO:5, and encodes the amino acid sequenceof SEQ ID NO:6. Genomic clone pJK1131 (denoted pJK1131 OrfC genomicclone, in the form of an E. coli plasmid vector containing “OrfC” genefrom Schizochytrium ATCC 20888) was deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______.The nucleotide sequence of pJK1131 OrfC genomic clone, and the aminoacid sequence encoded by this plasmid are encompassed by the presentinvention.

A genomic clone described herein as pBR002 OrfC genomic clone, isolatedfrom Schizochytrium sp. N230D, comprises, to the best of the presentinventors' knowledge, the nucleotide sequence of SEQ ID NO:5, andencodes the amino acid sequence of SEQ ID NO:6. Genomic clone pBR002(denoted pBR002 OrfC genomic clone, in the form of an E. coli plasmidvector containing the OrfC gene sequence from Schizochytrium sp. N230D)was deposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, andassigned ATCC Accession No. ______. The nucleotide sequence of pBR002OrfC genomic clone, and the amino acid sequence encoded by this plasmidare encompassed by the present invention.

OrfC was compared with known sequences in a standard BLAST search asdescribed above. At the nucleic acid level, OrfC has no significanthomology to any known nucleotide sequence. At the amino acid level(Blastp), the sequences with the greatest degree of homology to ORFCwere: Moritella marinus (Vibrio marinus) ORF11 (Accession No. ABO25342),which is 45% identical to ORFC over 514 amino acid residues, Shewanellasp. hypothetical protein 8 (Accession No. U73935), which is 49%identical to ORFC over 447 amino acid residues, Nostoc sp. hypotheticalprotein (Accession No. NC_(—)003272), which is 49% identical to ORFCover 430 amino acid residues, and Shewanella sp. hypothetical protein 7(Accession No. U73935), which is 37% identical to ORFC over 930 aminoacid residues.

The first domain in OrfC is a DH domain, also referred to herein asORFC-DH1. This is one of two DH domains in OrfC, and therefore isdesignated DH1. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 778 ofSEQ ID NO:5 (OrfC) to an ending point of between about positions 1233and 1350 of SEQ ID NO:5 (based on homology to other PUFA PKS domains,the position of the DH1 domain spans from about position 1 to aboutposition 1350; based on Pfam analysis, a DH core region spans from aboutposition 778 to about position 1233). The nucleotide sequence containingthe sequence encoding the ORFC-DH1 domain is represented herein as SEQID NO:27 (positions 1-1350 of SEQ ID NO:5). The amino acid sequencecontaining the DH1 domain spans from a starting point of between aboutpositions 1 and 260 of SEQ ID NO:6 (ORFC) to an ending point of betweenabout positions 411 and 450 of SEQ ID NO:6 (again, referring to theoverall homology to PUFA PKS DH domains and to Pfam core regions,respectively). The amino acid sequence containing the ORFC-DH1 domain isrepresented herein as SEQ ID NO:28 (positions 1-450 of SEQ ID NO:6).

The characteristics of both the DH domains (see below for DH 2) in thePUFA PKS systems have been described in the preceding sections. Thisclass of enzyme removes HOH from a β-keto acyl-ACP and leaves a transdouble bond in the carbon chain. The DH domains of the PUFA PKS systemsshow homology to bacterial DH enzymes associated with their FAS systems(rather than to the DH domains of other PKS systems). A subset ofbacterial DH's, the FabA-like DH's, possesses cis-trans isomeraseactivity (Heath et al., J. Biol. Chem., 271, 27795 (1996)). It is thehomologies to the FabA-like DH's that indicate that one or both of theDH domains is responsible for insertion of the cis double bonds in thePUFA PKS products.

The second domain in OrfC is a DH domain, also referred to herein asORFC-DH2. This is the second of two DH domains in OrfC, and therefore isdesignated DH2. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1351 and 2437of SEQ ID NO:5 (OrfC) to an ending point of between about positions 2607and 2847 of SEQ ID NO:5 (based on homology to other PUFA PKS domains,the position of the DH2 domain spans from about position 1351 to aboutposition 2845; based on Pfam analysis, a DH core region spans from aboutposition 2437 to about position 2847). The nucleotide sequencecontaining the sequence encoding the ORFC-DH2 domain is representedherein as SEQ ID NO:29 (positions 1351-2847 of SEQ ID NO:5). The aminoacid sequence containing the DH2 domain spans from a starting point ofbetween about positions 451 and 813 of SEQ ID NO:6 (ORFC) to an endingpoint of between about positions 869 and 949 of SEQ ID NO:6 (again,referring to the overall homology to PUFA PKS DH domains and to Pfamcore regions, respectively). The amino acid sequence containing theORFC-DH2 domain is represented herein as SEQ ID NO:30 (positions 451-949of SEQ ID NO:6). This DH domain comprises the amino acidsH-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) atpositions 426-440 of SEQ ID NO:30. DH biological activity has beendescribed above.

The third domain in OrfC is an ER domain, also referred to herein asORFC-ER. This domain is contained within the nucleotide sequencespanning from a starting point of about position 2995 of SEQ ID NO:5(OrfC) to an ending point of about position 4506 of SEQ ID NO:5. Thenucleotide sequence containing the sequence encoding the ORFC-ER domainis represented herein as SEQ ID NO:31 (positions 2995-4506 of SEQ IDNO:5). The amino acid sequence containing the ER domain spans from astarting point of about position 999 of SEQ ID NO:6 (ORFC) to an endingpoint of about position 1502 of SEQ ID NO:6. The amino acid sequencecontaining the ORFC-ER domain is represented herein as SEQ ID NO:32(positions 999-1502 of SEQ ID NO:6). ER biological activity has beendescribed above.

Accessory Proteins

According to the present invention, a domain or protein having4′-phosphopantetheinyl transferase (PPTase) biological activity(function) is characterized as the enzyme that transfers a4′-phosphopantetheinyl moiety from Coenzyme A to the acyl carrierprotein (ACP). This transfer to an invariant serine reside of the ACPactivates the inactive apo-form to the holo-form. In both polyketide andfatty acid synthesis, the phosphopantetheine group forms thioesters withthe growing acyl chains. The PPTases are a family of enzymes that havebeen well characterized in fatty acid synthesis, polyketide synthesis,and non-ribosomal peptide synthesis. The sequences of many PPTases areknown, and crystal structures have been determined (e.g., Reuter K,Mofid M R, Marahiel M A, Ficner R. “Crystal structure of the surfactinsynthetase-activating enzyme sfp: a prototype of the4′-phosphopantetheinyl transferase superfamily” EMBO J. 1999 Dec. 1;18(23):6823-31) as well as mutational analysis of amino acid residuesimportant for activity (Mofid M R, Finking R, Essen L O, Marahiel M A.“Structure-based mutational analysis of the 4′-phosphopantetheinyltransferases Sfp from Bacillus subtilis: carrier protein recognition andreaction mechanism” Biochemistry. 2004 Apr. 13; 43(14):4128-36).

The present inventors have identified two sequences (genes) in theArabidopsis whole genome database that are likely to encode PPTases.These sequences (GenBank Accession numbers; AAG51443 and AAC05345) arecurrently listed as encoding “Unknown Proteins”. They can be identifiedas putative PPTases based on the presence in the translated proteinsequences of several signature motifs including; G(I/V)D andWxxKE(A/S)xxK (SEQ ID NO:33), (listed in Lambalot et al., 1996 ascharacteristic of all PPTases). In addition, these two putative proteinscontain two additional motifs typically found in PPTases typicallyassociated with PKS and non-ribosomal peptide synthesis systems; i.e.,FN(I/L/V)SHS (SEQ ID NO:34) and (1N/L)G(I/L/V)D(I/L/V) (SEQ ID NO:35).Furthermore, these motifs occur in the expected relative positions inthe protein sequences. It is likely that homologues of the Arabidopsisgenes are present in other plants, such as tobacco. Again, these genescan be cloned and expressed to see if the enzymes they encode canactivate the Schizochytrium ORFA ACP domains, or alternatively, OrfAcould be expressed directly in the transgenic plant (either targeted tothe plastid or the cytoplasm).

Another heterologous PPTase which has been demonstrated by the inventorsto recognize the OrfA ACP domains described herein as substrates is theHet I protein of Nostoc sp. PCC 7120 (formerly called Anabaena sp. PCC7120).

One embodiment of the present invention relates to an isolated nucleicacid molecule comprising a nucleic acid sequence from a non-bacterialPUFA PKS system, a homologue thereof, a fragment thereof, and/or anucleic acid sequence that is complementary to any of such nucleic acidsequences. In one aspect, the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of: (a) a nucleic acid sequence encoding an aminoacid sequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, and biologically active fragments thereof; (b) anucleic acid sequence encoding an amino acid sequence selected from thegroup consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, and biologically active fragmentsthereof; (c) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to at least 500 consecutive aminoacids of said amino acid sequence of (a), wherein said amino acidsequence has a biological activity of at least one domain of apolyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; (d)a nucleic acid sequence encoding an amino acid sequence that is at leastabout 60% identical to said amino acid sequence of (b), wherein saidamino acid sequence has a biological activity of at least one domain ofa polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; or(e) a nucleic acid sequence that is fully complementary to the nucleicacid sequence of (a), (b), (c), or (d). In a further embodiment, nucleicacid sequences including a sequence encoding the active site domains orother functional motifs described above for several of the PUFA PKSdomains are encompassed by the invention.

According to the present invention, an amino acid sequence that has abiological activity of at least one domain of a PUFA PKS system is anamino acid sequence that has the biological activity of at least onedomain of the PUFA PKS system described in detail herein, as exemplifiedby the Schizochytrium PUFA PKS system. The biological activities of thevarious domains within the Schizochytrium PUFA PKS system have beendescribed in detail above. Therefore, an isolated nucleic acid moleculeof the present invention can encode the translation product of any PUFAPKS open reading frame, PUFA PKS domain, biologically active fragmentthereof, or any homologue of a naturally occurring PUFA PKS open readingframe or domain which has biological activity. A homologue of givenprotein or domain is a protein or polypeptide that has an amino acidsequence which differs from the naturally occurring reference amino acidsequence (i.e., of the reference protein or domain) in that at least oneor a few, but not limited to one or a few, amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). Preferred homologues of a PUFA PKSprotein or domain are described in detail below. It is noted thathomologues can include synthetically produced homologues, naturallyoccurring allelic variants of a given protein or domain, or homologoussequences from organisms other than the organism from which thereference sequence was derived.

In general, the biological activity or biological action of a protein ordomain refers to any function(s) exhibited or performed by the proteinor domain that is ascribed to the naturally occurring form of theprotein or domain as measured or observed in vivo (i.e., in the naturalphysiological environment of the protein) or in vitro (i.e., underlaboratory conditions). Biological activities of PUFA PKS systems andthe individual proteins/domains that make up a PUFA PKS system have beendescribed in detail elsewhere herein. Modifications of a protein ordomain, such as in a homologue or mimetic (discussed below), may resultin proteins or domains having the same biological activity as thenaturally occurring protein or domain, or in proteins or domains havingdecreased or increased biological activity as compared to the naturallyoccurring protein or domain. Modifications which result in a decrease inexpression or a decrease in the activity of the protein or domain, canbe referred to as inactivation (complete or partial), down-regulation,or decreased action of a protein or domain. Similarly, modificationswhich result in an increase in expression or an increase in the activityof the protein or domain, can be referred to as amplification,overproduction, activation, enhancement, up-regulation or increasedaction of a protein or domain. A functional domain of a PUFA PKS systemis a domain (i.e., a domain can be a portion of a protein) that iscapable of performing a biological function (i.e., has biologicalactivity).

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation), itsnatural milieu being the genome or chromosome in which the nucleic acidmolecule is found in nature. As such, “isolated” does not necessarilyreflect the extent to which the nucleic acid molecule has been purified,but indicates that the molecule does not include an entire genome or anentire chromosome in which the nucleic acid molecule is found in nature.An isolated nucleic acid molecule can include a gene. An isolatednucleic acid molecule that includes a gene is not a fragment of achromosome that includes such gene, but rather includes the codingregion and regulatory regions associated with the gene, but noadditional genes naturally found on the same chromosome. An isolatednucleic acid molecule can also include a specified nucleic acid sequenceflanked by (i.e., at the 5′ and/or the 3′ end of the sequence)additional nucleic acids that do not normally flank the specifiednucleic acid sequence in nature (i.e., heterologous sequences). Isolatednucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivativesof either DNA or RNA (e.g., cDNA). Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a protein or domainof a protein.

Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications provide the desired effect on PUFA PKS systembiological activity as described herein. Protein homologues (e.g.,proteins encoded by nucleic acid homologues) have been discussed indetail above.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, 1989). For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, PCR amplification and/ormutagenesis of selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

The minimum size of a nucleic acid molecule of the present invention isa size sufficient to form a probe or oligonucleotide primer that iscapable of forming a stable hybrid (e.g., under moderate, high or veryhigh stringency conditions) with the complementary sequence of a nucleicacid molecule useful in the present invention, or of a size sufficientto encode an amino acid sequence having a biological activity of atleast one domain of a PUFA PKS system according to the presentinvention. As such, the size of the nucleic acid molecule encoding sucha protein can be dependent on nucleic acid composition and percenthomology or identity between the nucleic acid molecule and complementarysequence as well as upon hybridization conditions per se (e.g.,temperature, salt concentration, and formamide concentration). Theminimal size of a nucleic acid molecule that is used as anoligonucleotide primer or as a probe is typically at least about 12 toabout 15 nucleotides in length if the nucleic acid molecules are GC-richand at least about 15 to about 18 bases in length if they are AT-rich.There is no limit, other than a practical limit, on the maximal size ofa nucleic acid molecule of the present invention, in that the nucleicacid molecule can include a sequence sufficient to encode a biologicallyactive fragment of a domain of a PUFA PKS system, an entire domain of aPUFA PKS system, several domains within an open reading frame (Orf) of aPUFA PKS system, an entire Orf of a PUFA PKS system, or more than oneOrf of a PUFA PKS system.

In one embodiment of the present invention, an isolated nucleic acidmolecule comprises or consists essentially of a nucleic acid sequenceencoding an amino acid sequence selected from the group of: SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, or biologically active fragmentsthereof. In one aspect, the nucleic acid sequence is selected from thegroup of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31.

In one embodiment of the present invention, any of the above-describedPUFA PKS amino acid sequences, as well as homologues of such sequences,can be produced with from at least one, and up to about 20, additionalheterologous amino acids flanking each of the C- and/or N-terminal endof the given amino acid sequence. The resulting protein or polypeptidecan be referred to as “consisting essentially of” a given amino acidsequence. According to the present invention, the heterologous aminoacids are a sequence of amino acids that are not naturally found (i.e.,not found in nature, in vivo) flanking the given amino acid sequence orwhich would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the given amino acid sequenceas it occurs in the gene, if such nucleotides in the naturally occurringsequence were translated using standard codon usage for the organismfrom which the given amino acid sequence is derived. Similarly, thephrase “consisting essentially of”, when used with reference to anucleic acid sequence herein, refers to a nucleic acid sequence encodinga given amino acid sequence that can be flanked by from at least one,and up to as many as about 60, additional heterologous nucleotides ateach of the 5′ and/or the 3′ end of the nucleic acid sequence encodingthe given amino acid sequence. The heterologous nucleotides are notnaturally found (i.e., not found in nature, in vivo) flanking thenucleic acid sequence encoding the given amino acid sequence as itoccurs in the natural gene.

The present invention also includes an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding an amino acid sequencehaving a biological activity of at least one domain of a PUFA PKSsystem. In one aspect, such a nucleic acid sequence encodes a homologueof any of the Schizochytrium PUFA PKS ORFs or domains, including: SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein the homologue has abiological activity of at least one (or two, three, four or more) domainof a PUFA PKS system as described previously herein.

In one aspect of the invention, a homologue of a Schizochytrium PUFA PKSprotein or domain encompassed by the present invention comprises anamino acid sequence that is at least about 60% identical to at least 500consecutive amino acids of an amino acid sequence chosen from: SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein said amino acid sequence hasa biological activity of at least one domain of a PUFA PKS system. In afurther aspect, the amino acid sequence of the homologue is at leastabout 60% identical to at least about 600 consecutive amino acids, andmore preferably to at least about 700 consecutive amino acids, and morepreferably to at least about 800 consecutive amino acids, and morepreferably to at least about 900 consecutive amino acids, and morepreferably to at least about 1000 consecutive amino acids, and morepreferably to at least about 1100 consecutive amino acids, and morepreferably to at least about 1200 consecutive amino acids, and morepreferably to at least about 1300 consecutive amino acids, and morepreferably to at least about 1400 consecutive amino acids, and morepreferably to at least about 1500 consecutive amino acids of any of SEQID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or to the full length of SEQ IDNO:6. In a further aspect, the amino acid sequence of the homologue isat least about 60% identical to at least about 1600 consecutive aminoacids, and more preferably to at least about 1700 consecutive aminoacids, and more preferably to at least about 1800 consecutive aminoacids, and more preferably to at least about 1900 consecutive aminoacids, and more preferably to at least about 2000 consecutive aminoacids of any of SEQ ID NO:2 or SEQ ID NO:4, or to the full length of SEQID NO:4. In a further aspect, the amino acid sequence of the homologueis at least about 60% identical to at least about 2100 consecutive aminoacids, and more preferably to at least about 2200 consecutive aminoacids, and more preferably to at least about 2300 consecutive aminoacids, and more preferably to at least about 2400 consecutive aminoacids, and more preferably to at least about 2500 consecutive aminoacids, and more preferably to at least about 2600 consecutive aminoacids, and more preferably to at least about 2700 consecutive aminoacids, and more preferably to at least about 2800 consecutive aminoacids, and even more preferably, to the full length of SEQ ID NO:2.

In another aspect, a homologue of a Schizochytrium PUFA PKS protein ordomain encompassed by the present invention comprises an amino acidsequence that is at least about 65% identical, and more preferably atleast about 70% identical, and more preferably at least about 75%identical, and more preferably at least about 80% identical, and morepreferably at least about 85% identical, and more preferably at leastabout 90% identical, and more preferably at least about 95% identical,and more preferably at least about 96% identical, and more preferably atleast about 97% identical, and more preferably at least about 98%identical, and more preferably at least about 99% identical to an aminoacid sequence chosen from: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,over any of the consecutive amino acid lengths described in theparagraph above, wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system.

In one aspect of the invention, a homologue of a Schizochytrium PUFA PKSprotein or domain encompassed by the present invention comprises anamino acid sequence that is at least about 60% identical to an aminoacid sequence chosen from: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein said amino acid sequencehas a biological activity of at least one domain of a PUFA PKS system.In a further aspect, the amino acid sequence of the homologue is atleast about 65% identical, and more preferably at least about 70%identical, and more preferably at least about 75% identical, and morepreferably at least about 80% identical, and more preferably at leastabout 85% identical, and more preferably at least about 90% identical,and more preferably at least about 95% identical, and more preferably atleast about 96% identical, and more preferably at least about 97%identical, and more preferably at least about 98% identical, and morepreferably at least about 99% identical to an amino acid sequence chosenfrom: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system.

According to the present invention, the term “contiguous” or“consecutive”, with regard to nucleic acid or amino acid sequencesdescribed herein, means to be connected in an unbroken sequence. Forexample, for a first sequence to comprise 30 contiguous (or consecutive)amino acids of a second sequence, means that the first sequence includesan unbroken sequence of 30 amino acid residues that is 100% identical toan unbroken sequence of 30 amino acid residues in the second sequence.Similarly, for a first sequence to have “100% identity” with a secondsequence means that the first sequence exactly matches the secondsequence with no gaps between nucleotides or amino acids.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches, blastn for nucleic acid searches, and blastX for nucleic acidsearches and searches of translated amino acids in all 6 open readingframes, all with standard default parameters, wherein the query sequenceis filtered for low complexity regions by default (described inAltschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.” Nucleic Acids Res.25:3389-3402, incorporated herein by reference in its entirety); (2) aBLAST 2 alignment (using the parameters described below); (3) and/orPSI-BLAST with the standard default parameters (Position-SpecificIterated BLAST). It is noted that due to some differences in thestandard parameters between BLAST 2.0 Basic BLAST and BLAST 2, twospecific sequences might be recognized as having significant homologyusing the BLAST 2 program, whereas a search performed in BLAST 2.0 BasicBLAST using one of the sequences as the query sequence may not identifythe second sequence in the top matches. In addition, PSI-BLAST providesan automated, easy-to-use version of a “profile” search, which is asensitive way to look for sequence homologues. The program firstperforms a gapped BLAST database search. The PSI-BLAST program uses theinformation from any significant alignments returned to construct aposition-specific score matrix, which replaces the query sequence forthe next round of database searching. Therefore, it is to be understoodthat percent identity can be determined by using any one of theseprograms.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

In another embodiment of the invention, an amino acid sequence havingthe biological activity of at least one domain of a PUFA PKS system ofthe present invention includes an amino acid sequence that issufficiently similar to a naturally occurring PUFA PKS protein orpolypeptide that a nucleic acid sequence encoding the amino acidsequence is capable of hybridizing under moderate, high, or very highstringency conditions (described below) to (i.e., with) a nucleic acidmolecule encoding the naturally occurring PUFA PKS protein orpolypeptide (i.e., to the complement of the nucleic acid strand encodingthe naturally occurring PUFA PKS protein or polypeptide). Preferably, anamino acid sequence having the biological activity of at least onedomain of a PUFA PKS system of the present invention is encoded by anucleic acid sequence that hybridizes under moderate, high or very highstringency conditions to the complement of a nucleic acid sequence thatencodes a protein comprising an amino acid sequence represented by anyof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

In another embodiment of the invention, a nucleotide sequence of thepresent invention is a nucleotide sequence isolated from (obtainablefrom), identical to, or a homologue of, the nucleotide sequence from aSchizochytrium, wherein the nucleotide sequence from a Schizochytrium(including either strand of a DNA molecule from Schizochytrium)hybridizes under moderate, high, or very high stringency conditions to anucleotide sequence encoding an amino acid sequence represented by anyof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. In one embodiment,the Schizochytrium is Schizochytrium ATCC 20888. In another embodiment,the Schizochytrium is a daughter strain of Schizochytrium 20888,including mutated strains thereof (e.g., N230D).

Methods to deduce a complementary sequence are known to those skilled inthe art.

It should be noted that since amino acid sequencing and nucleic acidsequencing technologies are not entirely error-free, the sequencespresented herein, at best, represent apparent sequences of PUFA PKSdomains and proteins of the present invention, or of the nucleotidesequences encoding such amino acid sequences.

As used herein, hybridization conditions refer to standard hybridizationconditions under which nucleic acid molecules are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., isincorporated by reference herein in its entirety (see specifically,pages 9.31-9.62). In addition, formulae to calculate the appropriatehybridization and wash conditions to achieve hybridization permittingvarying degrees of mismatch of nucleotides are disclosed, for example,in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al.,ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

Yet another embodiment of the present invention includes a nucleic acidmolecule comprising, consisting essentially of, or consisting of, anucleic acid sequence that is identical to, or that is a homologue of(as defined above) the nucleic acid sequence of a cDNA plasmid cloneselected from LIB3033-046-D2 (ATCC Accession No. ______), LIB3033-047-B5(ATCC Accession No. ______), or LIB81-042-B9 (ATCC Accession No.______). In another embodiment, the present invention includes a nucleicacid molecule comprising, consisting essentially of, or consisting of, anucleic acid sequence that is identical to, or that is a homologue of(as defined above) the nucleic acid sequence of a genomic plasmidselected from: pJK1126 (ATCC Accession No. ______), pJK1129 (ATCCAccession No. ______), pJK1131 (ATCC Accession No. ______), pJK306 (ATCCAccession No. ______), pJK320 (ATCC Accession No. ______), pJK324 (ATCCAccession No. ______), or pBR002 (ATCC Accession No. ______)

Yet another embodiment of the present invention includes a nucleic acidmolecule comprising, consisting essentially of, or consisting of, anucleic acid sequence that encodes an amino acid sequence that isidentical to, or that is a homologue of (as defined above) the aminoacid sequence encoded by a cDNA plasmid clone selected fromLIB3033-046-D2 (ATCC Accession No. ______), LIB3033-047-B5 (ATCCAccession No. ______), or LIB81-042-B9 (ATCC Accession No. ______). Inanother embodiment, the present invention includes a nucleic acidmolecule comprising, consisting essentially of, or consisting of, anucleic acid sequence that encodes an amino acid sequence that isidentical to, or that is a homologue of (as defined above) the aminoacid sequence encoded by a genomic plasmid selected from: pJK1126 (ATCCAccession No. ______), pJK1129 (ATCC Accession No. ______), pJK1131(ATCC Accession No. ______), pJK306 (ATCC Accession No. ______), pJK320(ATCC Accession No. ______), pJK324 (ATCC Accession No. ______), orpBR002 (ATCC Accession No. ______).

Another embodiment of the present invention includes a recombinantnucleic acid molecule comprising a recombinant vector and a nucleic acidmolecule comprising a nucleic acid sequence encoding an amino acidsequence having a biological activity of at least one domain or proteinof a PUFA PKS system as described herein. Such nucleic acid sequencesand domains or proteins are described in detail above. According to thepresent invention, a recombinant vector is an engineered (i.e.,artificially produced) nucleic acid molecule that is used as a tool formanipulating a nucleic acid sequence of choice and for introducing sucha nucleic acid sequence into a host cell. The recombinant vector istherefore suitable for use in cloning, sequencing, and/or otherwisemanipulating the nucleic acid sequence of choice, such as by expressingand/or delivering the nucleic acid sequence of choice into a host cellto form a recombinant cell. Such a vector typically containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid sequence to be clonedor delivered, although the vector can also contain regulatory nucleicacid sequences (e.g., promoters, untranslated regions) which arenaturally found adjacent to nucleic acid molecules of the presentinvention or which are useful for expression of the nucleic acidmolecules of the present invention (discussed in detail below). Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a plasmid. The vector can be maintained as anextrachromosomal element (e.g., a plasmid) or it can be integrated intothe chromosome of a recombinant organism (e.g., a microbe or a plant).The entire vector can remain in place within a host cell, or undercertain conditions, the plasmid DNA can be deleted, leaving behind thenucleic acid molecule of the present invention. The integrated nucleicacid molecule can be under chromosomal promoter control, under native orplasmid promoter control, or under a combination of several promotercontrols. Single or multiple copies of the nucleic acid molecule can beintegrated into the chromosome. A recombinant vector of the presentinvention can contain at least one selectable marker.

In one embodiment, a recombinant vector used in a recombinant nucleicacid molecule of the present invention is an expression vector. As usedherein, the phrase “expression vector” is used to refer to a vector thatis suitable for production of an encoded product (e.g., a protein ofinterest). In this embodiment, a nucleic acid sequence encoding theproduct to be produced (e.g., a PUFA PKS domain) is inserted into therecombinant vector to produce a recombinant nucleic acid molecule. Thenucleic acid sequence encoding the protein to be produced is insertedinto the vector in a manner that operatively links the nucleic acidsequence to regulatory sequences in the vector which enable thetranscription and translation of the nucleic acid sequence within therecombinant host cell.

In another embodiment, a recombinant vector used in a recombinantnucleic acid molecule of the present invention is a targeting vector. Asused herein, the phrase “targeting vector” is used to refer to a vectorthat is used to deliver a particular nucleic acid molecule into arecombinant host cell, wherein the nucleic acid molecule is used todelete or inactivate an endogenous gene within the host cell ormicroorganism (i.e., used for targeted gene disruption or knock-outtechnology). Such a vector may also be known in the art as a “knock-out”vector. In one aspect of this embodiment, a portion of the vector, butmore typically, the nucleic acid molecule inserted into the vector(i.e., the insert), has a nucleic acid sequence that is homologous to anucleic acid sequence of a target gene in the host cell (i.e., a genewhich is targeted to be deleted or inactivated). The nucleic acidsequence of the vector insert is designed to bind to the target genesuch that the target gene and the insert undergo homologousrecombination, whereby the endogenous target gene is deleted,inactivated or attenuated (i.e., by at least a portion of the endogenoustarget gene being mutated or deleted).

Typically, a recombinant nucleic acid molecule includes at least onenucleic acid molecule of the present invention operatively linked to oneor more transcription control sequences. As used herein, the phrase“recombinant molecule” or “recombinant nucleic acid molecule” primarilyrefers to a nucleic acid molecule or nucleic acid sequence operativelylinked to a transcription control sequence, but can be usedinterchangeably with the phrase “nucleic acid molecule”, when suchnucleic acid molecule is a recombinant molecule as discussed herein.According to the present invention, the phrase “operatively linked”refers to linking a nucleic acid molecule to a transcription controlsequence in a manner such that the molecule is able to be expressed whentransfected (i.e., transformed, transduced, transfected, conjugated orconduced) into a host cell. Transcription control sequences aresequences which control the initiation, elongation, or termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in a host cell or organism into which the recombinant nucleicacid molecule is to be introduced.

Recombinant nucleic acid molecules of the present invention can alsocontain additional regulatory sequences, such as translation regulatorysequences, origins of replication, and other regulatory sequences thatare compatible with the recombinant cell. In one embodiment, arecombinant molecule of the present invention, including those which areintegrated into the host cell chromosome, also contains secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed protein to be secreted from the cell that produces theprotein. Suitable signal segments include a signal segment that isnaturally associated with the protein to be expressed or anyheterologous signal segment capable of directing the secretion of theprotein according to the present invention. In another embodiment, arecombinant molecule of the present invention comprises a leadersequence to enable an expressed protein to be delivered to and insertedinto the membrane of a host cell. Suitable leader sequences include aleader sequence that is naturally associated with the protein, or anyheterologous leader sequence capable of directing the delivery andinsertion of the protein to the membrane of a cell.

The present inventors have found that the Schizochytrium PUFA PKS Orfs Aand B are closely linked in the genome and region between the Orfs hasbeen sequenced. The Orfs are oriented in opposite directions and 4244base pairs separate the start (ATG) codons (i.e. they are arranged asfollows: 3′OrfA5′-4244 bp-5′OrfB3′). Examination of the 4244 bpintergenic region did not reveal any obvious Orfs (no significantmatches were found on a BlastX search). Both Orfs A and B are highlyexpressed in Schizochytrium, at least during the time of oil production,implying that active promoter elements are embedded in this intergenicregion. These genetic elements are believed to have utility as abi-directional promoter sequence for transgenic applications. Forexample, in a preferred embodiment, one could clone this region, placeany genes of interest at each end and introduce the construct intoSchizochytrium (or some other host in which the promoters can be shownto function). It is predicted that the regulatory elements, under theappropriate conditions, would provide for coordinated, high levelexpression of the two introduced genes. The complete nucleotide sequencefor the regulatory region containing Schizochytrium PUFA PKS regulatoryelements (e.g., a promoter) is represented herein as SEQ ID NO:36.

In a similar manner, OrfC is highly expressed in Schizochytrium duringthe time of oil production and regulatory elements are expected toreside in the region upstream of its start codon. A region of genomicDNA upstream of OrfC has been cloned and sequenced and is representedherein as (SEQ ID NO:37). This sequence contains the 3886 nt immediatelyupstream of the OrfC start codon. Examination of this region did notreveal any obvious Orfs (i.e., no significant matches were found on aBlastX search). It is believed that regulatory elements contained inthis region, under the appropriate conditions, will provide forhigh-level expression of a gene placed behind them. Additionally, underthe appropriate conditions, the level of expression may be coordinatedwith genes under control of the A-B intergenic region (SEQ ID NO:36).

Therefore, in one embodiment, a recombinant nucleic acid molecule usefulin the present invention, as disclosed herein, can include a PUFA PKSregulatory region contained within SEQ ID NO:36 and/or SEQ ID NO:37.Such a regulatory region can include any portion (fragment) of SEQ IDNO:36 and/or SEQ ID NO:37 that has at least basal PUFA PKStranscriptional activity (at least basal promoter activity).

One or more recombinant molecules of the present invention can be usedto produce an encoded product (e.g., a PUFA PKS domain, protein, orsystem) of the present invention. In one embodiment, an encoded productis produced by expressing a nucleic acid molecule as described hereinunder conditions effective to produce the protein. A preferred method toproduce an encoded protein is by transfecting a host cell with one ormore recombinant molecules to form a recombinant cell. Suitable hostcells to transfect include, but are not limited to, any bacterial,fungal (e.g., yeast), insect, plant or animal cell that can betransfected. Host cells can be either untransfected cells or cells thatare already transfected with at least one other recombinant nucleic acidmolecule.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as algae, bacteria andyeast. In microbial systems, the term “transformation” is used todescribe an inherited change due to the acquisition of exogenous nucleicacids by the microorganism and is essentially synonymous with the term“transfection.” However, in animal cells, transformation has acquired asecond meaning which can refer to changes in the growth properties ofcells in culture after they become cancerous, for example. Therefore, toavoid confusion, the term “transfection” is preferably used with regardto the introduction of exogenous nucleic acids into animal cells, andthe term “transfection” will be used herein to generally encompasstransfection of animal cells, plant cells and transformation ofmicrobial cells, to the extent that the terms pertain to theintroduction of exogenous nucleic acids into a cell. Therefore,transfection techniques include, but are not limited to, transformation,particle bombardment, electroporation, microinjection, lipofection,adsorption, infection and protoplast fusion.

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transfectednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, integration of the nucleic acid molecules intoone or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals (e.g., ribosomebinding sites, Shine-Dalgarno sequences), modification of nucleic acidmolecules to correspond to the codon usage of the host cell, anddeletion of sequences that destabilize transcripts.

General discussion above with regard to recombinant nucleic acidmolecules and transfection of host cells is intended to be applied toany recombinant nucleic acid molecule discussed herein, including thoseencoding any amino acid sequence having a biological activity of atleast one domain from a PUFA PKS, those encoding amino acid sequencesfrom other PKS systems, and those encoding other proteins or domains.

This invention also relates to PUFA PKS systems (and proteins or domainsthereof) from microorganisms other than those described specificallyherein that are homologous in structure, domain organization and/orfunction to a Schizochytrium PUFA PKS system (and proteins or domainsthereof) as described herein. In one embodiment, the microorganism is anon-bacterial microorganism, and preferably, the microorganism is aeukaryotic microorganism. In addition, this invention relates to use ofthese microorganisms and the PUFA PKS systems or components thereof fromthese microorganisms in the various applications for a PUFA PKS system(e.g., genetically modified organisms and methods of producing bioactivemolecules) according to the present invention. Such microorganisms havethe following characteristics: (a) produces at least one PUFA; and (b)has an ability to produce increased PUFAs under dissolved oxygenconditions of less than about 5% of saturation in the fermentationmedium, as compared to production of PUFAs by said microorganism underdissolved oxygen conditions of greater than 5% of saturation, morepreferably 10% of saturation, more preferably greater than 15% ofsaturation and more preferably greater than 20% of saturation in thefermentation medium. A screening process for identification ofmicroorganisms comprising a PUFA PKS system is described in detail inU.S. Patent Application Publication No. 20020194641, supra. Theknowledge of the structure and function of the PUFA PKS proteins anddomains described herein, and the nucleotide sequence encoding the same,are useful tools for the identification, confirmation, and/or isolationof homologues of such proteins or polynucleotides.

According to the present invention, the term “Thraustochytrid” refers toany members of the order Thraustochytriales, which includes the familyThraustochytriaceae, and the term “Labyrinthulid” refers to any memberof the order Labyrinthulales, which includes the familyLabyrinthulaceae. The members of the family Labyrinthulaceae have beenconsidered to be members of the order Thraustochytriales, but inrevisions of the taxonomy of such organisms, the family is nowconsidered to be a member of the order Labyrinthulales, and bothLabyrinthulales and Thraustochytriales are considered to be members ofthe phylum Labyrinthulomycota.

Developments have resulted in frequent revision of the taxonomy of theThraustochytrids (thraustochytrids). Taxonomic theorists generally placeThraustochytrids with the algae or algae-like protists. However, becauseof taxonomic uncertainty, it would be best for the purposes of thepresent invention to consider the strains described in the presentinvention as Thraustochytrids to include the following organisms: Order:Thraustochytriales; Family: Thraustochytriaceae; Genera:Thraustochytrium (Species: sp., arudimentale, aureum, benthicola,globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum,roseum, striatum), Ulkenia (previously considered by some to be a memberof Thraustochytrium) (Species: sp., amoeboidea, kerguelensis, minuta,profunda, radiata, sailens, sarkariana, schizochytrops, visurgensis,yorkensis), Schizochytrium (Species: sp., aggregatum, limnaceum,mangrovei, minutum, octosporum), Japonochytrium (Species: sp., marinum),Aplanochytrium (Species: sp., haliotidis, kerguelensis, profunda,stocchinoi), Althornia (Species: sp., crouchii), or Dina (Species: sp.,marisalba, sinorifica).

Strains described in the present invention as Labyrinthulids include thefollowing organisms: Order: Labyrinthulales, Family: Labyrinthulaceae,Genera: Labyrinthula (Species: sp., algeriensis, coenocystis, chattonii,macrocystis, macrocystis atlantica, macrocystis macrocystis, marina,minuta, roscoffensis, valkanovii, vitellina, vitellina pacifica,vitellina vitellina, zopfii), Labyrinthuloides (Species: sp.,haliotidis, yorkensis), Labyrinthomyxa (Species: sp., marina),Diplophrys (Species: sp., archeri), Pyrrhosorus (Species: sp., marinus),Sorodiplophrys (Species: sp., stercorea) or Chlainydomyxa (Species: sp.,labyrinthuloides, montana) (although there is currently not a consensuson the exact taxonomic placement of Pyrrhosorus, Sorodiplophrys orChlamydomyxa).

It is recognized that at the time of this invention, revision in thetaxonomy of Thraustochytrids places the genus Labyrinthuloides in thefamily of Labyrinthulaceae and confirms the placement of the twofamilies Thraustochytriaceae and Labyrinthulaceae within theStramenopile lineage. It is noted that the Labyrinthulaceae aresometimes commonly called labyrinthulids or labyrinthula, orlabyrinthuloides and the Thraustochytriaceae are commonly calledthraustochytrids.

To produce significantly high yields of various bioactive moleculesusing the PUFA PKS system of the present invention, an organism,preferably a microorganism or a plant, can be genetically modified toaffect the activity of a PUFA PKS system. In one aspect, such anorganism can endogenously contain and express a PUFA PKS system, and thegenetic modification can be a genetic modification of one or more of thefunctional domains of the endogenous PUFA PKS system, whereby themodification has some effect on the activity of the PUFA PKS system. Inanother aspect, such an organism can endogenously contain and express aPUFA PKS system, and the genetic modification can be an introduction ofat least one exogenous nucleic acid sequence (e.g., a recombinantnucleic acid molecule), wherein the exogenous nucleic acid sequenceencodes at least one biologically active domain or protein from the sameor a second PKS system and/or a protein that affects the activity ofsaid PUFA PKS system (e.g., a phosphopantetheinyl transferases (PPTase),discussed below). In yet another aspect, the organism does notnecessarily endogenously (naturally) contain a PUFA PKS system, but isgenetically modified to introduce at least one recombinant nucleic acidmolecule encoding an amino acid sequence having the biological activityof at least one domain of a PUFA PKS system. In this aspect, PUFA PKSactivity is affected by introducing or increasing PUFA PKS activity inthe organism. Various embodiments associated with each of these aspectswill be discussed in greater detail below.

Therefore, according to the present invention, one embodiment relates toa genetically modified microorganism, wherein the microorganismexpresses a PKS system comprising at least one biologically activedomain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS)system. The at least one domain of the PUFA PKS system is encoded by anucleic acid sequence described herein. The genetic modification affectsthe activity of the PKS system in the organism. The genetically modifiedmicroorganism can include any one or more of the above-identifiednucleic acid sequences, and/or any of the other homologues of any of theSchizochytrium PUFA PKS ORFs or domains as described in detail above.

As used herein, a genetically modified microorganism can include agenetically modified bacterium, protist, microalgae, fungus, or othermicrobe, and particularly, any of the genera of the orderThraustochytriales (e.g., a Thraustochytrid) described herein. Such agenetically modified microorganism has a genome which is modified (i.e.,mutated or changed) from its normal (i.e., wild-type or naturallyoccurring) form such that the desired result is achieved (i.e.,increased or modified PUFA PKS activity and/or production of a desiredproduct using the PUFA PKS system or component thereof). Geneticmodification of a microorganism can be accomplished using classicalstrain development and/or molecular genetic techniques. Such techniquesknown in the art and are generally disclosed for microorganisms, forexample, in Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press. The reference Sambrook et al.,ibid., is incorporated by reference herein in its entirety. Agenetically modified microorganism can include a microorganism in whichnucleic acid molecules have been inserted, deleted or modified (i.e.,mutated; e.g., by insertion, deletion, substitution, and/or inversion ofnucleotides), in such a manner that such modifications provide thedesired effect within the microorganism.

Preferred microorganism host cells to modify according to the presentinvention include, but are not limited to, any bacteria, protist,microalga, fungus, or protozoa. In one aspect, preferred microorganismsto genetically modify include, but are not limited to, any microorganismof the order Thraustochytriales or any microorganism of the orderLabyrinthulales. Particularly preferred host cells for use in thepresent invention could include microorganisms from a genus including,but not limited to: Thraustochytrium, Ulkenia, Schizochytrium,Japonochytrium, Aplanochytrium, Althornia, Elina, Labyrinthula,Labyrinthuloides, Labyrinthomyxa, Diplophrys, Pyrrhosorus,Sorodiplophrys or Chlamydomyxa. Other examples of suitable hostmicroorganisms for genetic modification include, but are not limited to,yeast including Saccharomyces cerevisiae, Saccharomyces carlsbergensis,or other yeast such as Candida, Kluyveromyces, or other fungi, forexample, filamentous fungi such as Aspergillus, Neurospora, Penicillium,etc. Bacterial cells also may be used as hosts. This includesEscherichia coli, which can be useful in fermentation processes.Alternatively, a host such as a Lactobacillus species or Bacillusspecies can be used as a host.

Another embodiment of the present invention relates to a geneticallymodified plant or part of a plant (e.g., wherein the plant has beengenetically modified to express a PUPA PKS system described herein),which includes at least the core PUFA PKS enzyme complex and, in oneembodiment, at least one PUFA PKS accessory protein, (e.g., a PPTase),so that the plant produces PUFAs. Preferably, the plant is an oil seedplant, wherein the oil seeds or oil in the oil seeds contain PUFAsproduced by the PUFA PKS system. Such oils contain a detectable amountof at least one target or primary PUFA that is the product of the PUFAPKS system. Plants are not known to endogenously contain a PUFA PKSsystem, and therefore, the PUFA PKS systems of the present inventionrepresent an opportunity to produce plants with unique fatty acidproduction capabilities. It is a particularly preferred embodiment ofthe present invention to genetically engineer plants to produce one ormore PUFAs in the same plant, including, EPA, DHA, DPA, ARA, GLA, SDAand others. The present invention offers the ability to create any oneof a number of “designer oils” in various ratios and forms. Moreover,the disclosure of the PUFA PKS genes from the particular marineorganisms described herein offer the opportunity to more readily extendthe range of PUFA production and successfully produce such PUFAs withintemperature ranges used to grow most crop plants.

Methods for the genetic engineering of plants are well known in the art.For instance, numerous methods for plant transformation have beendeveloped, including biological and physical transformation protocols.See, for example, Miki et al., “Procedures for Introducing Foreign DNAinto Plants” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pp. 67-88. In addition, vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pp. 89-119.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. See, for example, Borsch et al., Science 227:1229 (1985).A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteriawhich genetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by numerous references, including Gruber et al., supra, Miki etal., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S.Pat. Nos. 4,940,838 and 5,464,763.

Another generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles. The expression vector is introduced intoplant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206(1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

As used herein, a genetically modified plant can include any geneticallymodified plant including higher plants and particularly, any consumableplants or plants useful for producing a desired bioactive molecule ofthe present invention. “Plant parts”, as used herein, include any partsof a plant, including, but not limited to, seeds (immature or mature),oils, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems,explants, etc. A genetically modified plant has a genome that ismodified (i.e., mutated or changed) from its normal (i.e., wild-type ornaturally occurring) form such that the desired result is achieved(e.g., PUFA PKS activity and production of PUFAs). Genetic modificationof a plant can be accomplished using classical strain development and/ormolecular genetic techniques. Methods for producing a transgenic plant,wherein a recombinant nucleic acid molecule encoding a desired aminoacid sequence is incorporated into the genome of the plant, are known inthe art. A preferred plant to genetically modify according to thepresent invention is preferably a plant suitable for consumption byanimals, including humans.

Preferred plants to genetically modify according to the presentinvention (i.e., plant host cells) include, but are not limited to anyhigher plants, including both dicotyledonous and monocotyledonousplants, and particularly consumable plants, including crop plants andespecially plants used for their oils. Such plants can include, forexample: canola, soybeans, rapeseed, linseed, corn, safflowers,sunflowers and tobacco. Other preferred plants include those plants thatare known to produce compounds used as pharmaceutical agents, flavoringagents, nutraceutical agents, functional food ingredients orcosmetically active agents or plants that are genetically engineered toproduce these compounds/agents.

According to the present invention, a genetically modified microorganismor plant includes a microorganism or plant that has been modified usingrecombinant technology. As used herein, genetic modifications thatresult in a decrease in gene expression, in the function of the gene, orin the function of the gene product (i.e., the protein encoded by thegene) can be referred to as inactivation (complete or partial),deletion, interruption, blockage or down-regulation of a gene. Forexample, a genetic modification in a gene which results in a decrease inthe function of the protein encoded by such gene, can be the result of acomplete deletion of the gene (i.e., the gene does not exist, andtherefore the protein does not exist), a mutation in the gene whichresults in incomplete or no translation of the protein (e.g., theprotein is not expressed), or a mutation in the gene which decreases orabolishes the natural function of the protein (e.g., a protein isexpressed which has decreased or no enzymatic activity or action).Genetic modifications that result in an increase in gene expression orfunction can be referred to as amplification, overproduction,overexpression, activation, enhancement, addition, or up-regulation of agene.

The genetic modification of a microorganism or plant according to thepresent invention preferably affects the activity of the PKS systemexpressed by the plant, whether the PKS system is endogenous andgenetically modified, endogenous with the introduction of recombinantnucleic acid molecules into the organism, or provided completely byrecombinant technology. According to the present invention, to “affectthe activity of a PKS system” includes any genetic modification thatcauses any detectable or measurable change or modification in the PKSsystem expressed by the organism as compared to in the absence of thegenetic modification. A detectable change or modification in the PKSsystem can include, but is not limited to: the introduction of PKSsystem activity into an organism such that the organism now hasmeasurable/detectable PKS system activity (i.e., the organism did notcontain a PKS system prior to the genetic modification), theintroduction into the organism of a functional domain from a differentPKS system than a PKS system endogenously expressed by the organism suchthat the PKS system activity is modified (e.g., a bacterial PUFA PKSdomain or a type I PKS domain is introduced into an organism thatendogenously expresses a non-bacterial PUFA PKS system), a change in theamount of a bioactive molecule produced by the PKS system (e.g., thesystem produces more (increased amount) or less (decreased amount) of agiven product as compared to in the absence of the geneticmodification), a change in the type of a bioactive molecule produced bythe PKS system (e.g., the system produces a new or different product, ora variant of a product that is naturally produced by the system), and/ora change in the ratio of multiple bioactive molecules produced by thePKS system (e.g., the system produces a different ratio of one PUFA toanother PUFA, produces a completely different lipid profile as comparedto in the absence of the genetic modification, or places various PUFAsin different positions in a triacylglycerol as compared to the naturalconfiguration). Such a genetic modification includes any type of geneticmodification and specifically includes modifications made by recombinanttechnology and by classical mutagenesis.

It should be noted that reference to increasing the activity of afunctional domain or protein in a PUFA PKS system refers to any geneticmodification in the organism containing the domain or protein (or intowhich the domain or protein is to be introduced) which results inincreased functionality of the domain or protein system and can includehigher activity of the domain or protein (e.g., specific activity or invivo enzymatic activity), reduced inhibition or degradation of thedomain or protein system, and overexpression of the domain or protein.For example, gene copy number can be increased, expression levels can beincreased by use of a promoter that gives higher levels of expressionthan that of the native promoter, or a gene can be altered by geneticengineering or classical mutagenesis to increase the activity of thedomain or protein encoded by the gene.

Similarly, reference to decreasing the activity of a functional domainor protein in a PUFA PKS system refers to any genetic modification inthe organism containing such domain or protein (or into which the domainor protein is to be introduced) which results in decreased functionalityof the domain or protein and includes decreased activity of the domainor protein, increased inhibition or degradation of the domain or proteinand a reduction or elimination of expression of the domain or protein.For example, the action of domain or protein of the present inventioncan be decreased by blocking or reducing the production of the domain orprotein, “knocking out” the gene or portion thereof encoding the domainor protein, reducing domain or protein activity, or inhibiting theactivity of the domain or protein. Blocking or reducing the productionof a domain or protein can include placing the gene encoding the domainor protein under the control of a promoter that requires the presence ofan inducing compound in the growth medium. By establishing conditionssuch that the inducer becomes depleted from the medium, the expressionof the gene encoding the domain or protein (and therefore, of proteinsynthesis) could be turned off. Blocking or reducing the activity ofdomain or protein could also include using an excision technologyapproach similar to that described in U.S. Pat. No. 4,743,546,incorporated herein by reference. To use this approach, the geneencoding the protein of interest is cloned between specific geneticsequences that allow specific, controlled excision of the gene from thegenome. Excision could be prompted by, for example, a shift in thecultivation temperature of the culture, as in U.S. Pat. No. 4,743,546,or by some other physical or nutritional signal.

In one embodiment of the present invention, a genetic modificationincludes a modification of a nucleic acid sequence encoding an aminoacid sequence that has a biological activity of at least one domain of anon-bacterial PUFA PKS system as described herein. Such a modificationcan be to an amino acid sequence within an endogenously (naturally)expressed non-bacterial PUFA PKS system, whereby a microorganism thatnaturally contains such a system is genetically modified by, forexample, classical mutagenesis and selection techniques and/or moleculargenetic techniques, include genetic engineering techniques. Geneticengineering techniques can include, for example, using a targetingrecombinant vector to delete a portion of an endogenous gene, or toreplace a portion of an endogenous gene with a heterologous sequence.Examples of heterologous sequences that could be introduced into a hostgenome include sequences encoding at least one functional domain fromanother PKS system, such as a different non-bacterial PUFA PKS system, abacterial PUFA PKS system, a type I PKS system, a type II PKS system, ora modular PKS system. Other heterologous sequences to introduce into thegenome of a host includes a sequence encoding a protein or functionaldomain that is not a domain of a PKS system, but which will affect theactivity of the endogenous PKS system. For example, one could introduceinto the host genome a nucleic acid molecule encoding aphosphopantetheinyl transferase (discussed below). Specificmodifications that could be made to an endogenous PUPA PKS system arediscussed in detail below.

In another aspect of this embodiment of the invention, the geneticmodification can include: (1) the introduction of a recombinant nucleicacid molecule encoding an amino acid sequence having a biologicalactivity of at least one domain of a non-bacterial PUFA PKS system;and/or (2) the introduction of a recombinant nucleic acid moleculeencoding a protein or functional domain that affects the activity of aPUFA PKS system, into a host. The host can include: (1) a host cell thatdoes not express any PKS system, wherein all functional domains of a PKSsystem are introduced into the host cell, and wherein at least onefunctional domain is from a non-bacterial PUFA PKS system; (2) a hostcell that expresses a PKS system (endogenous or recombinant) having atleast one functional domain of a non-bacterial PUFA PKS system, whereinthe introduced recombinant nucleic acid molecule can encode at least oneadditional non-bacterial PUFA PKS domain function or another protein ordomain that affects the activity of the host PKS system; and (3) a hostcell that expresses a PKS system (endogenous or recombinant) which doesnot necessarily include a domain function from a non-bacterial PUFA PKS,and wherein the introduced recombinant nucleic acid molecule includes anucleic acid sequence encoding at least one functional domain of anon-bacterial PUFA PKS system. In other words, the present inventionintends to encompass any genetically modified organism (e.g.,microorganism or plant), wherein the organism comprises at least onenon-bacterial PUFA PKS domain function (either endogenously or byrecombinant modification), and wherein the genetic modification has ameasurable effect on the non-bacterial PUFA PKS domain function or onthe PKS system when the organism comprises a functional PKS system.

Therefore, using the PUFA PKS systems of the present invention, genemixing can be used to extend the range of PUFA products (and ratiosthereof) to include EPA, DPA, DHA, ARA, GLA, SDA and others, as well asto produce a wide variety of bioactive molecules, including antibiotics,other pharmaceutical compounds, and other desirable products. The methodto obtain these bioactive molecules includes not only the mixing ofgenes from various organisms but also various methods of geneticallymodifying the non-bacterial PUFA PKS genes disclosed herein. Knowledgeof the genetic basis and domain structure of the non-bacterial PUFA PKSsystem of the present invention provides a basis for designing novelgenetically modified organisms which produce a variety of bioactivemolecules. Although mixing and modification of any PKS domains andrelated genes are contemplated by the present inventors, by way ofexample, various possible manipulations of the PUFA-PKS system arediscussed in U.S. Patent Application Publication No. 20020194641, U.S.Patent Application Publication No. 20040235127, and U.S. PatentApplication Publication No. 20050100995, supra with regard to geneticmodification and bioactive molecule production.

The comparison of the Schizochytrium PUFA PKS architecture (domainorganization) with other PUFA PKS system architecture illustratesnature's ability to alter domain order as well as incorporate newdomains to create novel end products. In addition, the genes can now bemanipulated in the laboratory to create new products. Proposed herein isthe manipulation of PUFA PKS systems in either a directed or random wayto influence the end products. For example, in a preferred embodiment,one could envision substituting one of the DH (FabA-like) domains of thePUFA-PKS system for a DH domain that did not posses isomerizationactivity, potentially creating a molecule with a mix of cis- andtrans-double bonds. The current products of the Schizochytrium PUFA PKSsystem are DHA and DPA (C22:5 ω6). If one manipulated the system toproduce C20 fatty acids, one would expect the products to be EPA and ARA(C20:4 ω6). This could provide a new source for ARA. One could alsosubstitute domains from related PUFA-PKS systems that produced adifferent DHA to DPA ratio, for example, by using genes fromThraustochytrium 23B.

Additionally, one could envision specifically altering one of the ERdomains (e.g. removing, or inactivating) in the Schizochytrium PUFA PKSsystem (other PUFA PKS systems described so far do not have two ERdomains) to affect the end product profile. Similar strategies could beattempted in a directed manner for each of the distinct domains of thePUFA-PKS proteins using more or less sophisticated approaches. Of courseone would not be limited to the manipulation of single domains. Finally,one could extend the approach by mixing domains from the PUFA-PKS systemand other PKS or FAS systems (e.g., type I, type II, type III) to createan entire range of new end products. For example, one could introducethe PUFA-PKS DH domains into systems that do not normally incorporatecis double bonds into their end products.

Accordingly, encompassed by the present invention are methods togenetically modify microbial or plant cells by: genetically modifying atleast one nucleic acid sequence in the organism that encodes an aminoacid sequence having the biological activity of at least one functionaldomain of a PUFA PKS system according to the present invention, and/orexpressing at least one recombinant nucleic acid molecule comprising anucleic acid sequence encoding such amino acid sequence. Variousembodiments of such sequences, methods to genetically modify anorganism, and specific modifications have been described in detailabove. Typically, the method is used to produce a particular geneticallymodified organism that produces a particular bioactive molecule ormolecules.

In one embodiment of the present invention, it is contemplated that amutagenesis program could be combined with a selective screening processto obtain bioactive molecules of interest. This would include methods tosearch for a range of bioactive compounds. This search would not berestricted to production of those molecules with cis double bonds. Themutagenesis methods could include, but are not limited to: chemicalmutagenesis, gene shuffling, switching regions of the genes encodingspecific enzymatic domains, or mutagenesis restricted to specificregions of those genes, as well as other methods.

For example, high throughput mutagenesis methods could be used toinfluence or optimize production of the desired bioactive molecule. Oncean effective model system has been developed, one could modify thesegenes in a high throughput manner. Utilization of these technologies canbe envisioned on two levels. First, if a sufficiently selective screenfor production of a product of interest (e.g., ARA) can be devised, itcould be used to attempt to alter the system to produce this product(e.g., in lieu of, or in concert with, other strategies such as thosediscussed above). Additionally, if the strategies outlined aboveresulted in a set of genes that did produce the product of interest, thehigh throughput technologies could then be used to optimize the system.For example, if the introduced domain only functioned at relatively lowtemperatures, selection methods could be devised to permit removing thatlimitation. In one embodiment of the invention, screening methods areused to identify additional non-bacterial organisms having novel PKSsystems similar to the PUFA PKS system of Schizochytrium, as describedherein (see above). Homologous PKS systems identified in such organismscan be used in methods similar to those described herein for theSchizochytrium, as well as for an additional source of genetic materialfrom which to create, further modify and/or mutate a PUFA PKS system forexpression in that microorganism, in another microorganism, or in ahigher plant, to produce a variety of compounds.

It is recognized that many genetic alterations, either random ordirected, which one may introduce into a native (endogenous, natural)PUFA PKS system, will result in an inactivation of enzymatic functions.A preferred embodiment of the invention includes a system to select foronly those modifications that do not block the ability of the PUFA PKSsystem to produce a product. For example, the FabB-strain of E. coli isincapable of synthesizing unsaturated fatty acids and requiressupplementation of the medium with fatty acids that can substitute forits normal unsaturated fatty acids in order to grow (see Metz et al.,2001, supra). However, this requirement (for supplementation of themedium) can be removed when the strain is transformed with a functionalPUFA-PKS system (i.e. one that produces a PUFA product in the E. colihost—see (Metz et al., 2001, supra, FIG. 2A). The transformedFabB-strain now requires a functional PUFA-PKS system (to produce theunsaturated fatty acids) for growth without supplementation. The keyelement in this example is that production of a wide range ofunsaturated fatty acid will suffice (even unsaturated fatty acidsubstitutes such as branched chain fatty acids). Therefore, in anotherpreferred embodiment of the invention, one could create a large numberof mutations in one or more of the PUFA PKS genes disclosed herein, andthen transform the appropriately modified FabB-strain (e.g. createmutations in an expression construct containing an ER domain andtransform a FabB-strain having the other essential domains on a separateplasmid—or integrated into the chromosome) and select only for thosetransformants that grow without supplementation of the medium (i.e.,that still possessed an ability to produce a molecule that couldcomplement the FabB-defect). Additional screens could be developed tolook for particular compounds (e.g. use of GC for fatty acids) beingproduced in this selective subset of an active PKS system. One couldenvision a number of similar selective screens for bioactive moleculesof interest.

In one embodiment of invention, a genetically modified organism has amodification that changes at least one product produced by theendogenous PKS system, as compared to a wild-type organism.

In one embodiment, a genetically modified organism has been modified bytransfecting the organism with a recombinant nucleic acid moleculeencoding a protein that regulates the chain length of fatty acidsproduced by the PUFA PKS system. For example, the protein that regulatesthe chain length of fatty acids produced by the PUFA PKS system can be achain length factor that directs the synthesis of C20 units or C22units.

In another embodiment, a genetically modified organism expresses a PUFAPKS system comprising a genetic modification in a domain selected fromthe group consisting of a domain encoding β-hydroxy acyl-ACP dehydrase(DH) and a domain encoding 3-ketoacyl-ACP synthase (KS), wherein themodification alters the ratio of long chain fatty acids produced by thePUFA PKS system as compared to in the absence of the modification. Inone aspect of this embodiment, the modification is selected from thegroup consisting of a deletion of all or a part of the domain, asubstitution of a homologous domain from a different organism for thedomain, and a mutation of the domain.

In another embodiment, a genetically modified organism expresses a PUFAPKS system comprising a modification in an enoyl-ACP reductase (ER)domain, wherein the modification results in the production of adifferent compound as compared to in the absence of the modification. Inone aspect of this embodiment, the modification is selected from thegroup consisting of a deletion of all or a part of the ER domain, asubstitution of an ER domain from a different organism for the ERdomain, and a mutation of the ER domain.

In one embodiment of the invention, the genetically modified organismproduces a polyunsaturated fatty acid (PUFA) profile that differs fromthe naturally occurring organism without a genetic modification.

Many other genetic modifications useful for producing bioactivemolecules will be apparent to those of skill in the art, given thepresent disclosure, and various other modifications have been discussedpreviously herein. The present invention contemplates any geneticmodification related to a PUFA PKS system as described herein whichresults in the production of a desired bioactive molecule.

As described above, in one embodiment of the present invention, agenetically modified microorganism or plant includes a microorganism orplant which has an enhanced ability to synthesize desired bioactivemolecules (products) or which has a newly introduced ability tosynthesize specific products (e.g., to synthesize a specificantibiotic). According to the present invention, “an enhanced ability tosynthesize” a product refers to any enhancement, or up-regulation, in apathway related to the synthesis of the product such that themicroorganism or plant produces an increased amount of the product(including any production of a product where there was none before) ascompared to the wild-type microorganism or plant, cultured or grown,under the same conditions. Methods to produce such genetically modifiedorganisms have been described in detail above. In one preferredembodiment, the present invention relates to a genetically modifiedplant or part of a plant (e.g., wherein the plant has been geneticallymodified to express a PUFA PKS system described herein), which includesat least the core PUFA PKS enzyme complex and, in one embodiment, atleast one PUFA PKS accessory protein, (e.g., a PPTase), so that theplant produces PUFAs. Preferably, the plant is an oil seed plant,wherein the oil seeds or oil in the oil seeds contain PUFAs produced bythe PUFA PKS system. Such oils contain a detectable amount of at leastone target or primary PUFA that is the product of the PUFA PKS system.

The present inventors demonstrate herein the production of PUFAs in aplant that has been genetically modified to express the genes encoding aPUFA PKS system from Schizochytrium of the present invention and a PUFAPKS accessory enzyme, 4′-phosphopantetheinyl transferase (PPTase). Theoils produced by these plants contain significant quantities of both DHA(docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid(C22:5, n-6), which are the predominant PUFAs (the primary PUFAs)produced by the Schizochytrium from which the PUFA PKS genes werederived. Significantly, oils from plants that produce PUFAs using thePUFA PKS pathway have a different fatty acid profile than plants thatare genetically engineered to produce the same PUFAs by the “standard”pathway described above. In particular, oils from plants that have beengenetically engineered to produce specific PUFAs by the PUFA PKS pathwayare substantially free of the various intermediate products and sideproducts that accumulate in oils that are produced as a result of theuse of the standard PUFA synthesis pathway. This characteristic isdiscussed in detail below.

More particularly, efforts to produce long chain PUFAs in plants by the“standard” pathway have all taken the same basic approach, which isdictated by this synthesis pathway. These efforts relied on modificationof the plants' endogenous fatty acids by introduction of genes encodingvarious elongases and desaturases. Plants typically produce 18 carbonfatty acids (e.g., oleic acid, linoleic acid, linolenic acid) via theType II fatty acid synthase (FAS) in its plastids. Often, a singledouble bond is formed while that fatty acid is attached to ACP, and thenthe oleic acid (18:1) is cleaved from the ACP by the action of anacyl-ACP thioesterase. The free fatty acid is exported from the plastidand converted to an acyl-CoA. The 18:1 can be esterified tophosphatidylcholine (PC) and up to two more cis double bonds can beadded. The newly introduced elongases can utilize substrates in theacyl-CoA pool to add carbons in two-carbon increments. Newly introduceddesaturases can utilize either fatty acids esterified to PC, or those inthe acyl-CoA pool, depending on the source of the enzyme. Oneconsequence of this scheme for long chain PUFA production, however, isthat intermediates or side products in the pathway accumulate, whichoften represent the majority of the novel fatty acids in the plant oil,rather than the target long chain PUFA.

For example, using the standard or classical pathway as described above,when the target PUFA product (i.e., the PUFA product that one istargeting for production, trying to produce, attempting to produce, byusing the standard pathway) is DHA or EPA, for example (e.g., producedusing elongases and desaturases that will produce the DHA or EPA fromthe products of the FAS system), a variety of intermediate products andside products will be produced in addition to the DHA or EPA, and theseintermediate or side products frequently represent the majority of theproducts produced by the pathway, or are at least present in significantamounts in the lipids of the production organism. Such intermediate andside products include, but are not limited to, fatty acids having fewercarbons and/or fewer double bonds than the target, or primary PUFA, andcan include unusual fatty acid side products that may have the samenumber of carbons as the target or primary PUFA, but which may havedouble bonds in unusual positions. By way of example, in the productionof EPA using the standard pathway (e.g., see U.S. Patent ApplicationPublication 2004/0172682), while the target PUFA of the pathway is EPA(i.e., due to the use of elongases and desaturases that specifically acton the products of the FAS system to produce EPA), the oils produced bythe system include a variety of intermediate and side productsincluding: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STAor SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3,n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA;20:3, n-9) and various other intermediate or side products, such as20:0; 20:1 (Δ5); 20:1 (Δ11): 20:2 (Δ8,11); 20:2 (Δ11,14); 20:3(Δ5,11,14); 20:3 (Δ11,14,17); mead acid (20:3; Δ5,8,11); or 20:4(Δ5,1,14,17). Intermediates of the system can also include long chainPUFAs that are not the target of the genetic modification (e.g., astandard pathway enzyme system for producing DHA can actually producemore EPA as an intermediate product than DHA).

In contrast, the PUFA PKS synthase of the present invention does notutilize the fatty acid products of FAS systems. Instead, it produces thefinal PUFA product (the primary PUFA product) from the same smallprecursor molecule that is utilized by FASB and elongases (malonyl-CoA).Therefore, intermediates in the synthesis cycle are not released in anysignificant amount, and the PUFA product (also referred to herein as theprimary PUFA product) is efficiently transferred to phospholipids (PL)and triacylglycerol (TAG) fractions of the lipids. Indeed, a PUFA PKSsystem may produce two target or primary PUFA products (e.g., the PUFAPKS system from Schizochytrium produces both DHA and DPA n-6 as primaryproducts), but DPA is not an intermediate in the pathway to produce DHA.Rather, each is a separate product of the same PUFA PKS system.Therefore, the PUFA PKS genes of the present invention are an excellentmeans of producing oils containing PUFAs, and particularly, LCPUFAs in aheterologous host, such as a plant, wherein the oils are substantiallyfree (defined below) of the intermediates and side products thatcontaminate oils produced by the “standard” PUFA pathway.

Therefore, it is an object of the present invention to produce, via thegenetic manipulation of plants as described herein, polyunsaturatedfatty acids and, by extension, oils obtained from such plants (e.g.,obtained from the oil seeds of such plants) comprising these PUFAs.Examples of PUFAs that can be produced by the present invention include,but are not limited to, DHA (docosahexaenoic acid (C22:6, n-3)), ARA(eicosatetraenoic acid or arachidonic acid (C20:4, n-6)), DPA(docosapentaenoic acid (C22:5, n-6 or n-3)), and EPA (eicosapentaenoicacid (C20:5, n-3)). The present invention allows for the production ofcommercially valuable lipids enriched in one or more desired (target orprimary) PUFAs by the present inventors' development of geneticallymodified plants through the use of the polyketide synthase system of thepresent invention, as well as components thereof, that produces PUFAs.

According to the present invention, reference to a “primary PUFA”,“target PUFA”, “intended PUFA”, or “desired PUFA” refers to theparticular PUFA or PUFAs that are the intended product of the enzymepathway that is used to produce the PUFA(s). For example, when usingelongases and desaturases to modify products of the FAS system in theclassical pathway for PUFA production, one can select particularcombinations of elongases and desaturases that, when used together, willproduce a target or desired PUFA (e.g., DHA or EPA). As discussed above,such target or desired PUFA produced by the standard pathway may notactually be a “primary” PUFA in terms of the amount of PUFA as apercentage of total fatty acids produced by the system, due to theformation of intermediates and side products that can actually representthe majority of products produced by the system. However, one may usethe term “primary PUFA” even in that instance to refer to the target orintended PUFA product produced by the elongases or desaturases used inthe system.

In contrast to the classical pathway for PUFA production, when using aPUFA PKS system, a given PUFA PKS system derived from a particularorganism (or created from combining proteins and domains from PUFA PKSsystems) will produce particular PUFA(s), such that selection of a PUFAPKS system from a particular organism will result in the production ofspecified target or primary PUFAs. For example, use of a PUFA PKS systemfrom Schizochytrium according to the present invention will result inthe production of DHA and DPAn-6 as the target or primary PUFAs.However, as discussed above, the use of various proteins and domainswith proteins and domains from other PUPA PKS systems or other PKSsystems (that produce bioactive molecules other than PUFAs) can becombined (“mixed and matched”) to result in the production of differentPUFA profiles.

When using a PUFA PKS system of the present invention, oils produced bythe organism, such as a plant, are substantially free of intermediate orside products that are not the target or primary PUFA products and thatare not naturally produced by the endogenous FAS system in the wild-typeorganism (e.g., wild-type plants produce some shorter or medium chainPUFAs, such as 18 carbon PUFAs, via the FAS system, but there will benew, or additional, fatty acids produced in the plant as a result ofgenetic modification with a PUFA PKS system). In other words, ascompared to the profile of total fatty acids from the wild-type plant(not genetically modified) or the parent plant used as a recipient forthe indicated genetic modification, the majority of additional fattyacids in the profile of total fatty acids produced by plants that havebeen genetically modified with the PUFA PKS system of the presentinvention (or a component thereof), comprise the target or intended PUFAproducts of the PUFA PKS system (i.e., the majority of additional fattyacids in the total fatty acids that are produced by the geneticallymodified plant are the target PUFA(s)).

According to the present invention, reference to “intermediate products”or “side products” of an enzyme system that produces PUFAs refers to anyproducts, and particularly, fatty acid products, that are produced bythe enzyme system as a result of the production of the target or primaryPUFA of the system. Intermediate and side products are particularlysignificant in the standard pathway for PUFA synthesis and aresubstantially less significant in the PUFA PKS pathway, as discussedabove. It is noted that a primary or target PUFA of one enzyme systemmay be an intermediate of a different enzyme system where the primary ortarget product is a different PUFA, and this is particularly true ofproducts of the standard pathway of PUFA production, since the PUFA PKSsystem of the present invention substantially avoids the production ofintermediates. For example, when using the standard pathway to produceEPA, fatty acids such as GLA, DGLA and SDA are produced as intermediateproducts in significant quantities (e.g., U.S. Patent ApplicationPublication 2004/0172682 illustrates this point). Similarly, and alsoillustrated by U.S. Patent Application Publication 2004/0172682, whenusing the standard pathway to produce DHA, in addition to the fattyacids mentioned above, ETA and EPA (notably the target PUFA in the firstexample above) are produced in significant quantities and in fact, maybe present in significantly greater quantities relative to the totalfatty acid product than the target PUFA itself. This latter point isshown in U.S. Patent Application Publication 2004/0172682, where a plantthat was engineered to produce DHA by the standard pathway produces moreEPA as a percentage of total fatty acids than DHA.

Furthermore, to be “substantially free” of intermediate or side productsof the system for synthesizing PUFAs, or to not have intermediate orside products present in substantial amounts, means that anyintermediate or side product fatty acids that are produced in thegenetically modified plant (and/or parts of plants and/or seed oilfraction) as a result of the enzyme system for producing PUFAS (i.e.,that are not produced by the wild-type plant or the parent plant used asa recipient for the indicated genetic modification), are present in aquantity that is less than about 10% by weight of the total fatty acidsproduced by the plant, and more preferably less than about 9%, and morepreferably less than about 8%, and more preferably less than about 7%,and more preferably less than about 6%, and more preferably less thanabout 5%, and more preferably less than about 4%, and more preferablyless than about 3%, and more preferably less than about 2%, and morepreferably less than about 1% by weight of the total fatty acidsproduced by the plant.

In a preferred embodiment, to be “substantially free” of intermediate orside products of the system for synthesizing PUFAs, or to not haveintermediate or side products present in substantial amounts, means thatany intermediate or side product fatty acids that are produced in thegenetically modified plant (and/or parts of plants and/or seed oilfraction) as a result of the enzyme system for producing PUFAS (i.e.,that are not produced by the wild-type plant or the parent plant used asa recipient for the indicated genetic modification), are present in aquantity that is less than about 10% by weight of the total additionalfatty acids produced by the plant (additional fatty acids being thosethat are not produced by the wild-type plant or the parent plant used asa recipient for the indicated genetic modification), and more preferablyless than about 9%, and more preferably less than about 8%, and morepreferably less than about 7%, and more preferably less than about 6%,and more preferably less than about 5%, and more preferably less thanabout 4%, and more preferably less than about 3%, and more preferablyless than about 2%, and more preferably less than about 1% of the totaladditional fatty acids produced by the plant. Therefore, in contrast tothe fatty acid profile of plants that have been genetically modified toproduce PUFAs via the standard pathway, the majority of fatty acidproducts resulting from the genetic modification with a PUFA PKS systemwill be the target or intended fatty acid products.

When the target product of a PUFA PKS system is a long chain PUFA, suchas DHA or DPA (n-6 or n-3) produced by the PUFA PKS system of theinvention described herein, intermediate products and side products thatare not present in substantial amounts in the total lipids of plantsgenetically modified with such PUFA PKS can include, but are not limitedto: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA;18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6),arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9)and various other intermediate or side products, such as 20:0; 20:1(Δ5); 20:1 (Δ11); 20:2 (Δ8,11); 20:2 (Δ11,14); 20:3 (Δ5,11,14); 20:3(Δ11,14,17); mead acid (20:3; Δ5,8,11); or 20:4 (Δ5,1,14,17). Inaddition, when the target product is a particular PUFA, such as DHA, theintermediate products and side products that are not present insubstantial amounts in the total lipids of the genetically modifiedplants also include other PUFAs, including other PUFAs that are anatural product of a different PUFA PKS system, such as EPA in thisexample. It is to be noted that the PUFA PKS system of the presentinvention can also be used, if desired, to produce as a target PUFA aPUFA that can include GLA, SDA or DGLA (referring to embodiments whereoils are produced using components of a PUFA PKS system describedherein).

Using the knowledge of the genetic basis and domain structure of thePUFA PKS system described herein, the present inventors have designedand produced constructs encoding such a PUFA PKS system and havesuccessfully produced transgenic plants expressing the PUFA PKS system.The transgenic plants produce oils containing PUFAs, and the oils aresubstantially free of intermediate products that accumulate in astandard PUFA pathway (see Example 3). The present inventors have alsodemonstrated the use of the constructs to produce PUFAs in anothereukaryote, yeast, as a proof-of-concept experiment prior to theproduction of the transgenic plants (see Example 2). The examplesdemonstrate that transformation of both yeast and plants with a PUFA PKSsystem that produces DHA and DPAn-6 as the target PUFAs produces both ofthese PUFAs as the primary additional fatty acids in the total fattyacids of the plant (i.e., subtracting fatty acids that are produced inthe wild-type plant), and in the yeast and further, that any other fattyacids that are not present in the fatty acids of the wild-type plant arevirtually undetectable. Specific characteristics of genetically modifiedplants and parts and oils thereof of the present invention are describedin detail elsewhere herein.

Accordingly, one embodiment of the present invention is a method toproduce desired bioactive molecules (also referred to as products orcompounds) by growing or culturing a genetically modified microorganismor a genetically modified plant of the present invention (described indetail above). Such a method includes the step of culturing in afermentation medium or growing in a suitable environment, such as soil,a microorganism or plant, respectively, that has a genetic modificationas described previously herein and in accordance with the presentinvention. In a preferred embodiment, method to produce bioactivemolecules of the present invention includes the step of culturing underconditions effective to produce the bioactive molecule a geneticallymodified organism that expresses a PKS system comprising at least onebiologically active domain of a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system as described herein.

In the method of production of desired bioactive compounds of thepresent invention, a genetically modified microorganism is cultured orgrown in a suitable medium, under conditions effective to produce thebioactive compound. An appropriate, or effective, medium refers to anymedium in which a genetically modified microorganism of the presentinvention, when cultured, is capable of producing the desired product.Such a medium is typically an aqueous medium comprising assimilablecarbon, nitrogen and phosphate sources. Such a medium can also includeappropriate salts, minerals, metals and other nutrients. Microorganismsof the present invention can be cultured in conventional fermentationbioreactors. The microorganisms can be cultured by any fermentationprocess which includes, but is not limited to, batch, fed-batch, cellrecycle, and continuous fermentation. Preferred growth conditions forpotential host microorganisms according to the present invention arewell known in the art. The desired bioactive molecules produced by thegenetically modified microorganism can be recovered from thefermentation medium using conventional separation and purificationtechniques. For example, the fermentation medium can be filtered orcentrifuged to remove microorganisms, cell debris and other particulatematter, and the product can be recovered from the cell-free supernatantby conventional methods, such as, for example, ion exchange,chromatography, extraction, solvent extraction, membrane separation,electrodialysis, reverse osmosis, distillation, chemical derivatizationand crystallization. Alternatively, microorganisms producing the desiredcompound, or extracts and various fractions thereof, can be used withoutremoval of the microorganism components from the product.

In the method for production of desired bioactive compounds of thepresent invention, a genetically modified plant is cultured in afermentation medium or grown in a suitable medium such as soil. Anappropriate, or effective, fermentation medium has been discussed indetail above. A suitable growth medium for higher plants includes anygrowth medium for plants, including, but not limited to, soil, sand, anyother particulate media that support root growth (e.g. vermiculite,perlite, etc.) or Hydroponic culture, as well as suitable light, waterand nutritional supplements which optimize the growth of the higherplant. The genetically modified plants of the present invention areengineered to produce significant quantities of the desired productthrough the activity of the PKS system that is genetically modifiedaccording to the present invention. The compounds can be recoveredthrough purification processes which extract the compounds from theplant. In a preferred embodiment, the compound is recovered byharvesting the plant. In this embodiment, the plant can be consumed inits natural state or further processed into consumable products.

Bioactive molecules, according to the present invention, include anymolecules (compounds, products, etc.) that have a biological activity,and that can be produced by a PKS system that comprises at least oneamino acid sequence having a biological activity of at least onefunctional domain of a non-bacterial PUFA PKS system as describedherein. Such bioactive molecules can include, but are not limited to: apolyunsaturated fatty acid (PUFA), an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, and a cholesterol loweringformulation. One advantage of the non-bacterial PUFA PKS system of thepresent invention is the ability of such a system to introducecarbon-carbon double bonds in the cis configuration, and moleculesincluding a double bond at every third carbon. This ability can beutilized to produce a variety of compounds.

With respect to microorganisms, preferably, bioactive compounds ofinterest are produced by the genetically modified microorganism in anamount that is greater than about 0.05%, and preferably greater thanabout 0.1%, and more preferably greater than about 0.25%, and morepreferably greater than about 0.5%, and more preferably greater thanabout 0.75%, and more preferably greater than about 1%, and morepreferably greater than about 2.5%, and more preferably greater thanabout 5%, and more preferably greater than about 10%, and morepreferably greater than about 15%, and even more preferably greater thanabout 20% of the dry weight of the microorganism. For lipid compounds,preferably, such compounds are produced in an amount that is greaterthan about 5% of the dry weight of the microorganism. For otherbioactive compounds, such as antibiotics or compounds that aresynthesized in smaller amounts, those strains possessing such compoundsat of the dry weight of the microorganism are identified as predictablycontaining a novel. PKS system of the type described above. In someembodiments, particular bioactive molecules (compounds) are secreted bythe microorganism, rather than accumulating. Therefore, such bioactivemolecules are generally recovered from the culture medium and theconcentration of molecule produced will vary depending on themicroorganism and the size of the culture.

Preferably, a genetically modified organism (e.g., microorganism orplant) of the invention produces one or more polyunsaturated fatty acidsincluding, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DPA(C22:5, n-6 or n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), ALA (C18:3,n-3), and/or SDA (C18:4, n-3)), and more preferably, one or more longchain fatty acids, including, but not limited to, EPA (C20:5, n-3), DHA(C22:6, n-3), DPA (C22:5, n-6 or n-3), or DTA (C22:4, n-6). In aparticularly preferred embodiment, a genetically modified organism ofthe invention produces one or more polyunsaturated fatty acidsincluding, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3),and/or DPA (C22:5, n-6 or n-3).

Preferably, a genetically modified organism of the invention produces atleast one PUFA (the target PUPA), wherein the total fatty acid profilein the organism (or a part of the organism that accumulates PUFAs, suchas mature seeds or oil from such seeds, if the organism is an oil seedplant), comprises a detectable amount of this PUFA or PUFAs. Preferably,the PUFA is at least a 20 carbon PUFA and comprises at least 3 doublebonds, and more preferably at least 4 double bonds, and even morepreferably, at least 5 double bonds. In one embodiment, the PUFA is aPUFA that is not naturally produced by the organism (i.e., the wild-typeorganism in the absence of genetic modification or the parent organismused as a recipient for the indicated genetic modification).

Preferably, the total fatty acid profile in the organism (or part of theorganism that accumulates PUFAs) comprises at least 0.1% of the targetPUFA(s) by weight of the total fatty acids, and more preferably at leastabout 0.2%, and more preferably at least about 0.3%, and more preferablyat least about 0.4%, and more preferably at least about 0.5%, and morepreferably at least about 1%, and more preferably at least about 2%, andmore preferably at least about 3%, and more preferably at least about4%, and more preferably at least about 5%, and more preferably at leastabout 10%, and more preferably at least about 15%, and more preferablyat least about 20%, and more preferably at least about 25%, and morepreferably at least about 30%, and more preferably at least about 35%,and more preferably at least about 40%, and more preferably at leastabout 45%, and more preferably at least about 50%, and more preferablyat least about 55%, and more preferably at least about 60%, and morepreferably at least about 65%, and more preferably at least about 70%,and more preferably at least about 75%, and more preferably more that75% of at least one polyunsaturated fatty acid (the target PUFA) byweight of the total fatty acids, or any percentage from 0.1% to 75%, orgreater than 75% (up to 100% or about 100%), in 0.1% increments, of thetarget PUFA(s). As generally used herein, reference to a percentageamount of PUFA production is by weight of the total fatty acids producedby the organism, unless otherwise stated (e.g., in some cases,percentage by weight is relative to the total fatty acids produced by anenzyme complex, such as a PUFA PKS system). In one embodiment, totalfatty acids produced by a plant are presented as a weight percent asdetermined by gas chromatography (GC) analysis of a fatty acid methylester (FAME) preparation.

As described above, it is an additional characteristic of the totalfatty acids produced by a plant (and/or parts of plants or seed oilfraction) that has been genetically modified to express a PUFA PKS ofthe present invention that these total fatty acids produced by the plantcomprise less than about 10% by weight of any fatty acids other than thetarget PUFA(s) that are produced by the enzyme complex that produces thetarget PUFA(s) (e.g., DHA and DPAn-6 are the target PUFAs if the entirePUFA PKS system of the invention is used). Preferably, any fatty acidsthat are produced by the enzyme complex that produces the target PUFA(s)other than the target PUFA(s) are present at less than about 9%, andmore preferably less than about 8%, and more preferably less than about7%, and more preferably less than about 6%, and more preferably lessthan about 5%, and more preferably less than about 4%, and morepreferably less than about 3%, and more preferably less than about 2%,and more preferably less than about 1% by weight of the total fattyacids produced by the plant.

In another embodiment, any fatty acids that are produced by the enzymecomplex that produces the target PUFA(s) other than the target PUFA(s)are present at less than about 10% by weight of the total fatty acidsthat are produced by the enzyme complex that produces the target PUFA(s)in the plant (i.e., this measurement is limited to those total fattyacids that are produced by the enzyme complex that produces the targetPUFAs), and more preferably less than about 9%, and more preferably lessthan about 8%, and more preferably less than about 7%, and morepreferably less than about 6%, and more preferably less than about 5%,and more preferably less than about 4%, and more preferably less thanabout 3%, and more preferably less than about 2%, and more preferablyless than about 1% by weight of the total fatty acids that are producedby the enzyme complex that produces the target PUFA(s) in the plant.

In another aspect of this embodiment of the invention, the total fattyacids produced by the plant (and/or parts of plants or seed oilfraction) contain less than (or do not contain any more than) 10% PUFAshaving 18 or more carbons by weight of the total fatty acids produced bythe plant, other than the target PUFA(s) or the PUFAs that are presentin the wild-type plant (not genetically modified) or the parent plantused as a recipient for the indicated genetic modification. In furtheraspects, the total fatty acids produced by the plant (and/or parts ofplants or seed oil fraction) contain less than 9% PUFAs having 18 ormore carbons, or less than 8% PUFAs having 18 or more carbons, or lessthan 7% PUFAs having 18 or more carbons, or less than 6% PUFAs having 18or more carbons, or less than 5% PUFAs having 18 or more carbons, orless than 4% PUFAs having 18 or more carbons, or less than 3% PUFAshaving 18 or more carbons, or less than 2% PUFAs having 18 or morecarbons, or less than 1% PUFAs having 18 or more carbons by weight ofthe total fatty acids produced by the plant, other than the targetPUFA(s) or the PUFAs that are present in the wild-type plant (notgenetically modified) or the parent plant used as a recipient for theindicated genetic modification.

In another aspect of this embodiment of the invention, the total fattyacids produced by the plant (and/or parts of plants or seed oilfraction) contain less than (or do not contain any more than) 10% PUFAshaving 20 or more carbons by weight of the total fatty acids produced bythe plant, other than the target PUFA(s) or the PUFAs that are presentin the wild-type plant (not genetically modified) or the parent plantused as a recipient for the indicated genetic modification. In furtheraspects, the total fatty acids produced by the plant (and/or parts ofplants or seed oil fraction) contain less than 9% PUFAs having 20 ormore carbons, or less than 8% PUFAs having 20 or more carbons, or lessthan 7% PUFAs having 20 or more carbons, or less than 6% PUFAs having 20or more carbons, or less than 5% PUFAs having 20 or more carbons, orless than 4% PUFAs having 20 or more carbons, or less than 3% PUFAshaving 20 or more carbons, or less than 2% PUFAs having 20 or morecarbons, or less than 1% PUFAs having 20 or more carbons by weight ofthe total fatty acids produced by the plant, other than the targetPUFA(s) or the PUFAs that are present in the wild-type plant (notgenetically modified) or the parent plant used as a recipient for theindicated genetic modification.

In one embodiment, the total fatty acids in the plant (and/or parts ofplants or seed oil fraction) contain less than about 10% by weight ofthe total fatty acids produced by the plant, and more preferably lessthan about 9%, and more preferably less than about 8%, and morepreferably less than about 7%, and more preferably less than about 6%,and more preferably less than about 5%, and more preferably less thanabout 4%, and more preferably less than about 3%, and more preferablyless than about 2%, and more preferably less than about 1% of a fattyacid selected from any one or more of: gamma-linolenic acid (GLA; 18:3,n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenicacid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6);eicosatrienoic acid (ETA; 20:3, n-9) and various other fatty acids, suchas 20:0; 20:1 (Δ5); 20:1 (Δ11); 20:2 (Δ8,11); 20:2 (Δ11,14); 20:3(Δ5,11,14); 20:3 (Δ11,14,17); mead acid (20:3; Δ5,8,11); or 20:4(Δ5,1,14,17).

In another embodiment, the fatty acids that are produced by the enzymesystem that produces the long chain PUFAs in the plant contain less thanabout 10% by weight of the total fatty acids produced by the plant, andmore preferably less than about 9%, and more preferably less than about8%, and more preferably less than about 7%, and more preferably lessthan about 6%, and more preferably less than about 5%, and morepreferably less than about 4%, and more preferably less than about 3%,and more preferably less than about 2%, and more preferably less thanabout 1% of a fatty acid selected from: gamma-linolenic acid (GLA; 18:3,n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenicacid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6);eicosatrienoic acid

(ETA; 20:3, n-9) and various other fatty acids, such as 20:0; 20:1 (Δ5);20:1 (Δ11); 20:2 (Δ8,11); 20:2 (Δ11,14); 20:3 (Δ5,11,14); 20:3(Δ11,14,17); mead acid (20:3; Δ5,8,11); or 20:4 (Δ5,1,14,17).

In another embodiment, the fatty acids that are produced by the enzymesystem that produces the long chain PUFAs in the plant contain less thanabout 10% by weight of the total fatty acids produced by the plant, andmore preferably less than about 9%, and more preferably less than about8%, and more preferably less than about 7%, and more preferably lessthan about 6%, and more preferably less than about 5%, and morepreferably less than about 4%, and more preferably less than about 3%,and more preferably less than about 2%, and more preferably less thanabout 1% of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3,n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAshaving 20 carbons and three carbon-carbon double bonds, and PUFAs having22 carbons and two or three carbon-carbon double bonds.

In another embodiment, the fatty acids that are produced by the enzymesystem that produces the long chain PUFAs in the plant contain less thanabout 10% by weight of the total fatty acids produced by the plant, andmore preferably less than about 9%, and more preferably less than about8%, and more preferably less than about 7%, and more preferably lessthan about 6%, and more preferably less than about 5%, and morepreferably less than about 4%, and more preferably less than about 3%,and more preferably less than about 2%, and more preferably less thanabout 1% of each of the following PUFAs: gamma-linolenic acid (GLA;18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds,PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAshaving 22 carbons and two or three carbon-carbon double bonds.

In another embodiment, the fatty acids that are produced by the enzymesystem that produces the long chain PUFAs in the plant contain less thanabout 10% by weight of the total fatty acids produced by the plant, andmore preferably less than about 9%, and more preferably less than about8%, and more preferably less than about 7%, and more preferably lessthan about 6%, and more preferably less than about 5%, and morepreferably less than about 4%, and more preferably less than about 3%,and more preferably less than about 2%, and more preferably less thanabout 1% of any one or more of the following PUFAs: gamma-linolenic acid(GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon doublebonds, PUFAs having 20 carbons and three carbon-carbon double bonds, andPUFAs having 22 carbons and two or three carbon-carbon double bonds.

In one aspect of this embodiment of the invention, a geneticallymodified plant produces at least two target PUFAs (e.g., DHA andDPAn-6), and the total fatty acid profile in the plant, or the part ofthe plant that accumulates PUFAs (including oils from the oil seeds),comprises a detectable amount of these PUFAs. In this embodiment, thePUFAs are preferably each at least a 20 carbon PUFA and comprise atleast 3 double bonds, and more preferably at least 4 double bonds, andeven more preferably, at least 5 double bonds. Such PUFAs are mostpreferably chosen from DHA, DPAn-6 and EPA. In one aspect, the plantproduces DHA and DPAn-6 (the products of a PUFA PKS system describedherein), and the ratio of DHA to DPAn-6 is from about 1:10 to about10:1, including any ratio in between. In a one embodiment, the ratio ofDHA to DPA is from about 1:1 to about 3:1, and in another embodiment,about 2.5:1.

In another aspect of this embodiment of the invention, the plantproduces the total fatty acid profile represented by FIG. 5.

The invention further includes any seeds produced by the plantsdescribed above, as well as any oils produced by the plants or seedsdescribed above. The invention also includes any products produced usingthe plants, seed or oils described herein.

One embodiment of the present invention relates to a method to modify anendproduct containing at least one fatty acid, comprising adding to saidendproduct an oil produced by a recombinant host cell that expresses atleast one recombinant nucleic acid molecule comprising a nucleic acidsequence encoding at least one biologically active domain of a PUFA PKSsystem as described herein.

Preferably, the endproduct is selected from the group consisting of afood, a dietary supplement, a pharmaceutical formulation, a humanizedanimal milk, and an infant formula. Suitable pharmaceutical formulationsinclude, but are not limited to, an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, and a cholesterol loweringformulation. In one embodiment, the endproduct is used to treat acondition selected from the group consisting of: chronic inflammation,acute inflammation, gastrointestinal disorder, cancer, cachexia, cardiacrestenosis, neurodegenerative disorder, degenerative disorder of theliver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmunedisease, preeclampsia, preterm birth, age related maculopathy, pulmonarydisorder, and peroxisomal disorder.

Suitable food products include, but are not limited to, fine bakerywares, bread and rolls, breakfast cereals, processed and unprocessedcheese, condiments (ketchup, mayonnaise, etc.), dairy products (milk,yogurt), puddings and gelatine desserts, carbonated drinks, teas,powdered beverage mixes, processed fish products, fruit-based drinks,chewing gum, hard confectionery, frozen dairy products, processed meatproducts, nut and nut-based spreads, pasta, processed poultry products,gravies and sauces, potato chips and other chips or crisps, chocolateand other confectionery, soups and soup mixes, soya based products(milks, drinks, creams, whiteners), vegetable oil-based spreads, andvegetable-based drinks.

Yet another embodiment of the present invention relates to a method toproduce a humanized animal milk. This method includes the steps ofgenetically modifying milk-producing cells of a milk-producing animalwith at least one recombinant nucleic acid molecule comprising a nucleicacid sequence encoding at least one biologically active domain of a PUFAPKS system as described herein.

Methods to genetically modify a host cell and to produce a geneticallymodified non-human, milk-producing animal, are known in the art.Examples of host animals to modify include cattle, sheep, pigs, goats,yaks, etc., which are amenable to genetic manipulation and cloning forrapid expansion of a transgene expressing population. For animals,PKS-like transgenes can be adapted for expression in target organelles,tissues and body fluids through modification of the gene regulatoryregions. Of particular interest is the production of PUFAs in the breastmilk of the host animal.

Each publication or reference cited herein is incorporated herein byreference in its entirety.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example demonstrates that SchizochytriumOrfs A, B and Cencode a functional DHA/DPA synthesis enzyme via functional expressionin E. coli.

General Preparation of E. coli Transformants

The three genes encoding the Schizochytrium PUFA PKS system that produceDHA and DPA (Orfs A, B & C; SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5,respectively) were cloned into a single E. coli expression vector(derived from pET21c (Novagen)). The genes are transcribed as a singlemessage (by the T7 RNA-polymerase), and a ribosome-binding site clonedin front of each of the genes initiates translation. Modification of theOrf B coding sequence was needed to obtain production of a full-lengthOrf B protein in E. coli (see below). An accessory gene, encoding aPPTase (see below) was cloned into a second plasmid (derived frompACYC184, New England Biolabs).

The Orf B gene is predicted to encode a protein with a mass of ˜224 kDa.Initial attempts at expression of the gene in E. coli resulted inaccumulation of a protein with an apparent molecular mass of ˜165 kDa(as judged by comparison to proteins of known mass during SDS-PAGE).Examination of the Orf B nucleotide sequence revealed a regioncontaining 15 sequential serine codons—all of them being the TCT codon.The genetic code contains 6 different serine codons, and three of theseare used frequently in E. coli. The inventors used four overlappingoligonucleotides in combination with a polymerase chain reactionprotocol to resynthesize a small portion of the Orf B gene (a ˜195 basepair, BspHI to SacII restriction enzyme fragment) that contained theserine codon repeat region. In the synthetic Orf B fragment, a randommixture of the 3 serine codons commonly used by E. coli was used, andsome other potentially problematic codons were changed as well (i.e.,other codons rarely used by E. coli). The BspHI to SacII fragmentpresent in the original Orf B was replaced by the resynthesized fragment(to yield Orf B*) and the modified gene was cloned into the relevantexpression vectors. The modified OrfB* still encodes the amino acidsequence of SEQ ID NO:4. Expression of the modified Orf B* clone in E.coli resulted in the appearance of a ˜224 kDa protein, indicating thatthe full-length product of OrfB was produced. The sequence of theresynthesized Orf B* BspHI to SacII fragment is represented herein asSEQ ID NO:38. Referring to SEQ ID NO:38, the nucleotide sequence of theresynthesized BspHI to SacII region of Orf B is shown. The BspHIrestriction site and the SacII restriction site are identified. TheBspHI site starts at nucleotide 4415 of the Orf B CDS (SEQ ID NO:3)(note: there are a total of three BspHI sites in the Orf B CDS, whilethe SacII site is unique).

The ACP domains of the Orf A protein (SEQ ID NO:2 in Schizochytrium)must be activated by addition of phosphopantetheine group in order tofunction. The enzymes that catalyze this general type of reaction arecalled phosphopantetheine transferases (PPTases). E. coli contains twoendogenous PPTases, but it was anticipated that they would not recognizethe Orf A ACP domains from Schizochytrium. This was confirmed byexpressing Orfs A, B* (see above) and C in E. coli without an additionalPPTase. In this transformant, no DHA production was detected. Theinventors tested two heterologous PPTases in the E. coli PUFA PKSexpression system: (1) sfp (derived from Bacillus subtilis) and (2) HetI (from the cyanobacterium Nostoc strain 7120).

The sfp PPTase has been well characterized and is widely used due to itsability to recognize a broad range of substrates. Based on publishedsequence information (Nakana, et al., 1992, Molecular and GeneralGenetics 232: 313-321), an expression vector for sfp was built bycloning the coding region, along with defined up- and downstreamflanking DNA sequences, into a pACYC-184 cloning vector.Oligonucleotides were used to amplify the region of interest fromgenomic B. subtilus DNA. The oligonucleotides:

(forward; SEQ ID NO: 39) CGGGGTACCCGGGAGCCGCCTTGGCTTTGT; and(reverse; SEQ ID NO: 40) AAACTGCAGCCCGGGTCCAGCTGGCAGGCACCCTG,were used to amplify the region of interest from genomic B. subtilusDNA. Convenient restriction enzyme sites were included in theoligonucleotides to facilitate cloning in an intermediate, high copynumber vector and finally into the EcoRV site of pACYC184 to create theplasmid: pBR301. Examination of extracts of E. coli transformed withthis plasmid revealed the presence of a novel protein with the mobilityexpected for sfp. Co-expression of the sfp construct in cells expressingthe Orf A, B*, C proteins, under certain conditions, resulted in DHAproduction. This experiment demonstrated that sfp was able to activatethe SchizochytriumOrf A ACP domains. In addition, the regulatoryelements associated with the sfp gene were used to create an expressioncassette into which other genes could be inserted. Specifically, the sfpcoding region (along with three nucleotides immediately upstream of theATG) in pBR301 was replaced with a 53 base pair section of DNA designedso that it contains several unique (for this construct) restrictionenzyme sites. The initial restriction enzyme site in this region isNdeI. The ATG sequence embedded in this site is utilized as theinitiation methionine codon for introduced genes. The additionalrestriction sites (BglLL, Nod, SmaI, PmelI, HindIII, SpeI and XhoI) wereincluded to facilitate the cloning process. The functionality of thisexpression vector cassette was tested by using PCR to generate a versionof sfp with a NdeI site at the 5′ end and an XhoI site ate the 3′ end.This fragment was cloned into the expression cassette and transferredinto E. coli along with the Orf A, B* and C expression vector. Underappropriate conditions, these cells accumulated DHA, demonstrating thata functional sfp had been produced.

Het I is present in a cluster of genes in Nostoc known to be responsiblefor the synthesis of long chain hydroxy-fatty acids that are a componentof a glyco-lipid layer present in heterocysts of that organism (Blackand Wolk, 1994, J. Bacteriol. 176, 2282-2292; Campbell et al., 1997,Arch. Microbial. 167, 251-258). Het I activates the ACP domains of aprotein, Hgl E, present in that cluster. The two ACP domains of Hgl Ehave a high degree of sequence homology to the ACP domains found inSchizochytriumOrf A. A Het I expression construct was made using PCR.Specifically, SEQ ID NO:41 represents the amino acid sequence of theNostoc Het I protein. The endogenous start codon of Het I has not beenidentified (there is no methionine present in the putative protein).There are several potential alternative start codons (e.g., TTG and ATT)near the 5′ end of the open reading frame. No methionine codons (ATG)are present in the sequence. A Het I expression construct was made byusing PCR to replace the furthest 5′ potential alternative start codon(TTG) with a methionine codon (ATG, as part of the above described NdeIrestriction enzyme recognition site), and introducing an XhoI site atthe 3′ end of the coding sequence. The modified HetI coding sequence wasthen inserted into the NdeI and XhoI sites of the pACYC184 vectorconstruct containing the sfp regulatory elements. Expression of this HetI construct in E. coli resulted in the appearance of a new protein ofthe size expected from the sequence data. Co-expression of Het I withSchizochytriumOrfs A, B*, C in E. coli under several conditions resultedin the accumulation of DHA and DPA in those cells. In all of theexperiments in which sfp and Het I were compared, more DHA and DPAaccumulated in the cells containing the Het I construct than in cellscontaining the sfp construct.

Production of DHA and DPA in E. Coli Transformants

The two plasmids encoding: (1) the Schizochytrium PUFA PKS genes (OrfsA, B* and C) and (2) the PPTase (from sfp or from Het I) weretransformed into E. coli strain BL21 which contains an inducible T7 RNApolymerase gene. Synthesis of the Schizochytrium proteins was induced byaddition of IPTG to the medium, while PPTase expression was controlledby a separate regulatory element (see above). Cells were grown undervarious defined conditions and using either of the two heterologousPPTase genes. The cells were harvested and the fatty acids wereconverted to methyl-esters (FAME) and analyzed using gas-liquidchromatography.

Under several conditions, DHA and DPA were detected in E. coli cellsexpressing the Schizochytrium PUFA PKS genes, plus either of the twoheterologous PPTases (data not shown). No DHA or DPA was detected inFAMEs prepared from control cells (i.e., cells transformed with aplasmid lacking one of the Oils). The ratio of DHA to DPA observed in E.coli approximates that of the endogenous DHA and DPA production observedin Schizochytrium. The highest level of PUFA (DHA plus DPA),representing ˜17% of the total FAME, was found in cells grown at 32° C.in 765 medium (recipe available from the American Type CultureCollection) supplemented with 10% (by weight) glycerol. PUFAaccumulation was also observed when cells were grown in Luria Brothsupplemented with 5 or 10% glycerol, and when grown at 20° C. Selectionfor the presence of the respective plasmids was maintained by inclusionof the appropriate antibiotics during the growth, and IPTG (to a finalconcentration of 0.5 mM) was used to induce expression of Orfs A, B* andC. Co-expression of Het I or sfp with SchizochytriumOrfs A, B*, C in E.coli under several conditions resulted in the accumulation of DHA andDPA in those cells. In all of the experiments in which sfp and Het Iwere compared, more DHA and DPA accumulated in the cells containing theHet I construct than in cells containing the sfp construct.

Example 2

The following example shows the expression of genes encoding theSchizochytrium PUFA synthase (sOrfA, sOrfB and native Orf C) along withHet I in baker's yeast (Saccharomyces cerevisiae).

The Schizochytrium PUFA synthase genes and Het I were expressed in yeastusing materials obtained from Invitrogen (Invitrogen Corporation,Carlsbad, Calif.). The INVsc1 strain of Saccharomyces cerevisiae wasused along with the following transformation vectors: pYESLeu (sOrfA),pYES3/CT (sOrfB), pYES2/CT (OrfC) and pYESHis (HetI). To accommodateyeast codon useage, the nucleotide sequences for OrfA (SEQ ID NO:1) andfor OrfB (SEQ ID NO:3) were resynthesized. The nucleotide sequence forthe resynthesized OrfA (contained in pYESLeu), designated sOrfA, isrepresented herein by SEQ ID NO:43. SEQ ID NO:43 still encodes the OrfAamino acid sequence of SEQ ID NO:2. The nucleotide sequence for theresynthesized OrfB (contained in pYES3/CT), designated sOrfB, isrepresented herein by SEQ ID NO:44. SEQ ID NO:44 still encodes the OrfBamino acid sequence of SEQ ID NO:4. The OrfC nucleotide sequence used inthese experiments (contained in pYES2/CT) is the wild-type OrfC,represented by SEQ ID NO:5, and encoding SEQ ID NO:6.

Some of the vectors were modified to accommodate specific cloningrequirements (e.g., restriction sites for cloning). Appropriateselection media were used (as specified by Invitrogen), depending on theparticular experiment. The genes were cloned, in each case, behind aGAL1 promoter and expression was induced by re-suspension of washedcells in media containing galactose according to guidelines provide byInvitrogen. Cells were grown at 30° C. and harvested (by centrifugation)after being transferred to the induction medium. The cell pellets werefreeze dried and FAMEs were prepared using acidic methanol, extractedinto hexane and analyzed by GC.

A comparison of the fatty acid profile from yeast cells expressing theSchizochytrium PUFA synthase system (sOrfA, sOrfB, OrfC and Het I) andone obtained from control cells (lacking the sOrfA gene) collected ˜20hrs after induction, showed that two novel FAME peaks have appeared inthe profile of the strain expressing the complete PUFA synthase system(FIG. 3). These two peaks were identified as DPAn-6 and DHA bycomparison of the elution time with authentic standards and subsequentlyby MS analyses. As predicted from the characterization of theSchizochytrium PUPA synthase, aside from DHA and DPAn-6, no other novelpeaks are evident in the profile. FIG. 4 shows the region of the GCchromatogram of FIG. 3 which contains the PUFA FAMEs. Both the controlcells and the cell expressing the PUFA synthase contain a peak thatelutes near the DHA FAME. This has been identified as C26:0 FAME and(based on literature references) is derived from sphingolipids. Althoughit elutes close to the DHA peak, the resolution is sufficient so that itdoes not interfere with the quantitation of DHA. The DPA n-6 peak iswell separated from other endogenous yeast lipids in the FAME profile.In this particular example, the cells expressing the Schizochytrium PUFAsynthase system accumulated 2.4% DHA and 2.0% DPA n-6 (as a percentageof the total FAMEs). The sum of DHA and DPA n-6=4.4% of the measuredfatty acids in the cells. The ratio of DHA to DPA n-6 observed in thecells was ˜1.2:1.

Example 3

The following examples describes the expression of genes encoding theSchizochytrium PUFA synthase (Orf A, Orf B* and Orf C) along with Het Iin Arabidopsis.

The Schizochytrium Orfs A, B* (see Example 1) and C along with Het Iwere cloned (separately or in various combinations including all 4 geneson one Super-construct) into the appropriate binary vectors forintroduction of the genes into plants. Each gene was cloned behind alinin promoter and was followed by a linin terminator sequence(Chaudhary et al., 2001; PCT Publication Number No. WO 01/16340 A1). Forlocalization of the PUFA synthase in the cytoplasm of plant cells, noadditional protein encoding sequences were appended to the 5′ end of theOrfs. For directing the proteins to the plastid, additional 5′ sequencesencoding a plastid targeting sequence derived from a Brassica napusacyl-ACP thioesterase were added to the Orfs. The amino acid sequence ofthe encoded targeting peptide is: MLKLSCNVTNHLHTFSFFSDSSLFIPVNRRTLAVS(SEQ ID NO:42). The nucleotide sequences encoding this peptide wereplaced in frame with the start methionine codons of each PUFA synthaseOrf as well as the start codon of Het I.

More specifically, for one experiment described herein, the constructsand plants were prepared as follows:

Construction of pSBS4107: Acyl-ACP Transit Peptide-HetI: Acyl-ACPTransit Peptide-ORFC

This plant binary vector contained a double expression cassette whichtargeted the co-expression of HetI (SEQ ID NO:41) and ORFC (SEQ ID NO:6)to the plastid. The first expression cassette began with a signalpeptide (SEQ ID NO:42) derived from an acyl-ACP thioesterase gene fromBrassica juncea (GenBank Accession No. AJ294419) to target expression ofthe polypeptides to the plastid. The signal peptide was synthesized fromtwo overlapping oligos with an engineered AfiIII site at the 5′ end andan NcoI/Swal/XmaI multiple cloning site at the 3′ end. Immediatelydownstream was a sequence encoding for PPTase from Nostoc, encoded byHetI, to enable DHA to bind phosphopantetheine attachment sites. Thesecond expression cassette also began with the acyl-ACP signal peptidefollowed immediately in-frame with a cDNA encoding ORFC (SEQ ID NO:5).

The backbone of this plasmid, pSBS4055, was based on the plant binaryvector, pPZP200, described by Hajdukiewicz et al. (Plant MolecularBiology, 1994, 25:989-994). In place of the described multiple cloningsite, a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684), was inserted between the left and rightborder sequences. In addition to this cassette, two separate Lininpromoter/terminators in tandem from Linum usitatissumum (Flax orLinseed) (Chaudhary et al., 2001; PCT Publication Number No. WO 01/16340A1) were used to drive expression of ACP-HetI and ACP-ORFC. Standardrestriction cloning was used to fuse the synthetic Acyl-ACP signalpeptide in-frame with cDNAs encoding for either HetI or ORFC usingNcoI/XmaI and NcoI/SwaI restriction endonuclease sites, respectively, tothe 3′ end of the Linin promoter. The result was plasmid pSBS4107: a DNAsequence encoding the Acyl-ACP signal peptide-HetI and Acyl-ACP signalpeptide-ORFC polypeptides being placed in a binary vector underexpression control of the linin promoter/terminator. The linin promotercontrols the specific-temporal and tissue-specific expression of thetransgene during seed development. The Acyl-ACP signal peptide targetsthe expression of the protein to the plastid (Loader et al., 1993, PlantMol Biol 23:769-778). The complete plasmid map with annotated elementsis shown in FIG. 6.

Construction of pSBS5720: Acyl-ACP Transit Peptide-ORFB

This plant binary vector contained an expression cassette which targetedthe expression of ORFB (SEQ ID NO:4) to the plastid. Again, theexpression cassette began with a signal peptide derived from an acyl-ACPthioesterase gene from Brassica juncea (SEQ ID NO:42) to targetexpression of the polypeptide to the plastid. The signal peptide wassynthesized as above. Immediately downstream was a cDNA sequenceencoding for ORFB (SEQ ID NO:3, except for the resynthesized BspHI toSacII region of Orf B, represented by SEQ ID NO:38; see Example 1above).

The backbone of this plasmid, pSBS4055, was based on the plant binaryvector, pPZP200, described by Hajdukiewicz et al. (Plant MolecularBiology, 1994, 25:989-994). In place of the described multiple cloningsite, a phosphomannose isomerase (PMI) gene conferring host plantpositive selection for mannose-6-phosphate driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684), was inserted between the left and rightborder sequences. In addition to this cassette, a Lininpromoter/terminator from Linum usitatissumum (Flax or Linseed)(Chaudhary et al., 2001; PCT Publication Number WO 01/16340 A1) was usedto drive expression of ACP-ORFB. Standard restriction cloning was usedto fuse the synthetic Acyl-ACP signal peptide in-frame with cDNAsencoding for ORFB, to the 3′ end of the Linin promoter. The result wasplasmid pSBS5720: a DNA sequence encoding the Acyl-ACP signalpeptide-ORFB polypeptide being placed in a binary vector underexpression control of the linin promoter/terminator. The linin promotercontrols the specific-temporal and tissue-specific expression of thetransgene during seed development. The Acyl-ACP signal peptide targetsthe expression of the protein to the plastid (Loader et al., 1993, PlantMol Biol 23:769-778). The complete plasmid map with annotated elementsis shown in FIG. 7.

Construction of pSBS4757: Acyl-ACP Transit Peptide-ORFA

This plant binary vector contained an expression cassette which targetedthe expression of ORFA (SEQ ID NO:2) to the plastid. Again theexpression cassette began with a signal peptide derived from an acyl-ACPthioesterase gene from Brassica juncea (SEQ ID NO:42) to targetexpression of the polypeptide to the plastid. The signal peptide wassynthesized as above. Immediately downstream was a cDNA sequenceencoding for ORFA (SEQ ID NO:1).

The backbone of this plasmid, pSBS4055, was based on the plant binaryvector, pPZP200, described by Hajdukiewicz et al. (Plant MolecularBiology, 1994, 25:989-994). In place of the described multiple cloningsite, a neomycin phosphotransferase (nptII) gene conferring host plantKanamycin resistance driven by the mannopine synthasepromoter/terminator, was inserted between the left and right bordersequences. In addition, a Linin promoter/terminator from Linumusitatissumum (Flax or Linseed) (Chaudhary et al., 2001; PCT PublicationNumber WO 01/16340 A1) were used to drive expression of ACP-ORFA.Standard restriction cloning was used to fuse the synthetic Acyl-ACPsignal peptide in-frame with a cDNA encoding for ORFA to the 3′ end ofthe Linin promoter. The result was plasmid pSBS4757: a DNA sequenceencoding the Acyl-ACP signal peptide-ORFA polypeptide being placed in abinary vector under expression control of the linin promoter/terminator.The linin promoter controls the specific-temporal and tissue-specificexpression of the transgene during seed development. The Acyl-ACP signalpeptide targets the expression of the protein to the plastid (Loader etal., 1993, Plant Mol Biol 23:769-778). The complete plasmid map withannotated elements is shown in FIG. 8.

Standard methods were used for introduction of the genes intoArabidopsis (floral dipping into suspension of Agrobacterium strainscontaining the appropriate vectors; see Clough and Bent, Plant J.;16(6):735-43, 1998 December). Seeds obtained from those plants wereplated on selective medium and allowed to germinate. Some of the plantsthat grew were taken to maturity and the seeds analyzed for PUFAcontent. Based on PUFA content some of those seeds were taken forward tothe next generation. Pooled seeds obtained from those plants wereanalyzed for their fatty acid content. Analysis of a plant linetransformed with the constructs specifically described above and denoted269 is described in more detail below.

The top panel of FIG. 5 shows the typical fatty acid profile of wildtype Arabidopsis seeds as represented by GC separation and FID detectionof FAMEs prepared from a pooled seed sample. The predominant fatty acidsof wild type Arabidopsis seeds as represented by GC separation and FIDdetection of FAMEs prepared from a pooled seed sample are: 16:0, 18:0,16:1, 18:1, 20:1, 20:2 and 22:1. No DHA or DPA n-6 are present in thesamples from wild type seed. The lower panel of FIG. 5 shows the fattyacid profile of a pooled seed sample from one of the transgenicArabidopsis lines (line 269) expressing the Schizochytrium PUFA synthasegenes and Het I gene. The proteins expressed from these transgenescontain plastid targeting sequences. Two FAME peaks are present in theprofile from the transgenic plant seeds that are not present in theprofile from wild type seeds. The elution pattern of these two peaksexactly corresponds to the elution of authentic DHA and DPA n-6 (usingFAMEs prepared from Schizochytrium oil as standards, as well as acommercially purchased DHA standard from NuCheck Prep). In thisparticular example, the DHA peak represents 0.8% of total calculatedFAMEs while the DPA n-6 peak represents 1.7%. The sum of novel PUFAs is2.5% of total FAMEs. The appearance of DHA and DPA n-6 in the seed fattyacid profile demonstrates that introduced Schizochytrium PUFA synthasesystem functions when expressed in the plant cell and the proteins aretargeted to the plastid.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1-16. (canceled)
 17. An isolated nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of: a) anucleic acid sequence of SEQ ID NO:29; b) a nucleic acid sequenceencoding an amino acid sequence of SEQ ID NO:30; and c) a nucleic acidsequence encoding an amino acid sequence that is at least 90% identicalto SEQ ID NO:30 or that is a fragment of SEQ ID NO:30, wherein the aminoacid sequence has FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity,and wherein said amino acid sequence comprises the sequence ofH-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) atpositions corresponding to amino acids 426-440 of SEQ ID NO:30. 18-45.(canceled)
 46. A plant or a part of the plant, wherein the total fattyacid profile in the plant or part of the plant comprises detectableamounts of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA(docosapentaenoic acid (C22:5, n 6).
 47. The plant or a part of theplant of claim 46, wherein the ratio of DPAn-6 to DHA is less than 1:1.48. The plant or a part of the plant of claim 46, wherein the totalfatty acid profile in the plant or part of the plant contains less than5% by weight in total of all of the following PUFAs: gamma-linolenicacid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbondouble bonds, PUFAs having 20 carbons and three carbon-carbon doublebonds, and PUFAs having 22 carbons and two or three carbon-carbon doublebonds.
 49. The plant or a part of the plant of claim 46, wherein thetotal fatty acid profile in the plant or part of the plant comprises atleast about 0.5% by weight of at least one polyunsaturated fatty acid(PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6(C22:5n-6), and wherein the total fatty acid profile in the plant orpart of the plant contains less than 5% in total of all of the followingPUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbonsand four carbon-carbon double bonds, PUFAs having 20 carbons and threecarbon-carbon double bonds, and PUFAs having 22 carbons and two or threecarbon-carbon double bonds.
 50. The plant or a part of the plant ofclaim 46, wherein the total fatty acid profile in the plant or part ofthe plant comprises at least about 0.5% by weight of at least onepolyunsaturated fatty acid (PUFA) selected from the group consisting ofDHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acidprofile in the plant or part of the plant contains PUFAs having 18carbons and four carbon-carbon double bonds, PUFAs having 20 carbons andthree carbon-carbon double bonds, and PUFAs having 22 carbons and two orthree carbon-carbon double bonds.
 51. The plant or a part of the plantof claim 46, wherein the total fatty acid profile in the plant or partof the plant comprises at least about 0.5% by weight of at least onepolyunsaturated fatty acid (PUFA) selected from the group consisting ofDHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acidprofile in the plant or part of the plant contains less than 2% ofgamma-linolenic acid (GLA; 18:3, n-6) and dihomo-gamma-linolenic acid(DGLA or HGLA; 20:3, n-6).
 52. Seeds obtained from the plant or part ofthe plant of claim
 46. 53. A food product comprising the seeds of claim52.
 54. An oil obtained from seeds of the plant of claim 46 any.
 55. Afood product comprising the oil of claim
 54. 56. An oil blend comprisingthe oil of claim 54 and another oil. 57-59. (canceled)
 60. The oil ofclaim 54, comprising the following fatty acids: DHA (C22:6n-3), DPAn-6(C22:5n-6), oleic acid (C18:1), linolenic acid (C18:3), linoleic acid(C18:2), C16:0, C18:0, C20:0, C20:1n-9, C20:2n-6, C22:1n-9; wherein theoil comprises less than 0.5% of any of the following fatty acids:gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and fourcarbon-carbon double bonds, PUFAs having 20 carbons and threecarbon-carbon double bonds, and PUFAs having 22 carbons and two or threecarbon-carbon double bonds. 61-66. (canceled)
 67. The plant or a part ofthe plant of claim 46, wherein the ratio of DPAn-6 to DHA is 1:1 orgreater than 1:1.